THE EGGS OF MAMMALS
EXPERIMENTAL BIOLOGY SERIES
Editors: Philip Bard, Johns Hopkins University; L. R. Blinks,
Stanford University; W. B. Cannon, Harvard University; W. J.
Crozier, Harvard University; J. B. Collip, McGill University;
Hallowell Davis, Harvard University; S. R. Detwiler, Columbia
University; Selig Hecht, Columbia University; Hudson Hoagland,
Clark University; J. H. Northrop, Rockefeller Institute for Medical
Research; G. H. Parker, Harvard University; Gregory Pincus.
Harvard University; L. J. Stadler, The University of Missouri;
Sewall Wright, University of Chicago.
PACEMAKERS IN RELATION TO ASPECTS
OF BEHAVIOR. By Hudson Hoagland
NEUROEMBRYOLOGY. By Samuel R. Det-
wiler
THE EGGS OF MAMMALS. By Gregory
Pincus
Other volumes to follow
^ 1-
THE EGGS OF MAMMALS
BY
GREGORY PINCUS
Assistant Professor of General Physiology
Harvard University
NEW YORK
THE MACMILLAN COMPANY
1936
Copyright, 1936,
By the MACMILLAN" COMPANY
ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE
REPRODUCED IN ANY FORM WITHOUT PERMISSION IN WRITING
FROM THE PUBLISHER, EXCEPT BY A REVIEWER WHO WISHES
TO QUOTE BRIEF PASSAGES IN CONNECTION WITH A REVIEW
WRITTEN FOR INCLUSION IN MAGAZINE OR NEWSPAPER
Published, August, 1936
SET UP AND ELECTROTYPED BY T. MOREY * SON
PRINTED IN THE UNITED STATES OF AMERICA
This Book Is Dedicated to
W. E. Castle and W. J. Crazier
PREFACE
I should like to express my appreciation to Dr. J. B. Collip,
Dr. H. Selye, Dr. D. L. Thomson, and Dr. W. J. Crozier for
their kindness in reading the manuscript of this book before
publication. Their comments have been taken advantage
of in a manner for which I, not they, am responsible. I am
indebted too to Dr. F. H. A. Marshall and Mr. John Ham-
mond of Cambridge University for encouragement and
interest which led to the undertaking of this monograph,
and to my friend and collaborator Dr. E. V. Enzmann who
actively assisted in a number of the investigations herein
described. The National Research Council Committee for
Problems of Sex and the Josiah Macy Jr. Foundation pro-
vided grants making possible most of my own work, and
the preparation of the monograph itself is due in no small
measure to their assistance. To the editors and publishers
of the following journals I am indebted for permission to
reprint the various tables and figures indicated in the text:
the American Journal of Anatomy, the American Journal
of Physiology, the Anatomical Record, Archives de Biologic,
the Biological Bulletin, the Carnegie Institution of Wash-
ington Publications in Embryology, the Journal of Anatomy,
the Journal of Experimental Biology, the Journal of Experi-
mental Medicine, the Journal of Experimental Zoology, the
Journal of Morphology, the Quarterly Review of Biology,
and the Proceedings of the Royal Society.
I ask the understanding of the reader if this account of
the development of mammalian eggs seems at times to deal
in summary fashion with some of the voluminous literature
on this subject. The investigative aspects are what interest
and intrigue me. I emerge confessedly with the impression
that at best a qualitative basis for future work has been
estabhshed, and since I am possessed by the belief that
viii PREFACE
accurate quantitative observatioDs afford the means for
elucidating the nature of biological processes, I feel that this
is a book of interrogation, not explanation. If it does indeed
create curiosity its major objective will be attained.
Gregory Pincus
Cambridge, Mass.
July, 1936.
TABLE OF CONTENTS
PAGE
Preface vu
CHAPTER
I. Introduction 1
II. The Origin of the Definitive Ova 5
III. The Growth of the Ovum 32
IV. The Development and Atresia of Full-Grown Ova and
the Problem of Ovarian Parthenogenesis ... 42
V. Methods Employed in the Experimental ]\Ianipula-
tion of ^Mammalian Ova 62
VI. The Tubal History of Unfertilized Eggs .... 68
VIL Fertilization and Cleavage 75
VIII. The Activation of Unfertilized Eggs 98
IX. The Growth and Implantation of the Blastodermic
Vesicle 112
X. Summary and Recapitulation 128
Bibliography 131
Author Index 155
Subject Index 159
IX
THE EGGS OF MAMMALS
CHAPTER I
INTRODUCTION
The behavior of mammahan eggs from the time of their
genesis in the ovary to their implantation in the uterus is
the subject matter of this book. The attempt has been made
to include experimental investigations of the growth and
development of ova rather than morphological descriptions.
This is not an easy task, because an acute morphologist
may make deductions about the nature of his material
which are far more illuminating than those of an eager but
inexpert experimenter. Furthermore, except for certain
notable investigations of ovarian dynamics, there has been
no extensive inquiry into the physiology of living mam-
malian ova. It has been tacitly assumed, for example, that
the reactions involved in the activation of non-mammalian
ova occur also in mammalian eggs. Until quite recently no
attempt has been made to test even this assumption. Since
the middle of the last century a controversy has raged
about the possibility of ovarian parthenogenesis. Almost
every observer of mammalian ovaries has contributed an
opinion, but no one has tried to see if ovarian eggs can be
induced to develop parthenogenetically. Experimentation
has lagged presumably because of the difficulty of handling
living ova.
It is interesting to note that the discovery of the mamima-
lian egg by von Baer in 1827 led initially to extensive ob-
servations of living ova. At first the exact morphology of
the egg and its membranes was a matter of some debate
(see Wagner, 1836; Jones, 1837, 1838, 1885; Barry, 1838;
1
2 THE EGGS OF MAMMALS
Bischoff, 1842). Following Barry's (1839) initial observation
of cytoplasmic cleavage there ensued a long series of ob-
servations on the developmental history of fertilized eggs.
Attention gradually shifted from living eggs to fixed speci-
mens, chiefly employed for the determination of the exact
cytology of fertilization and the histological changes occur-
ring during differentiation. This resulted in the publication
of numerous detailed descriptions of the early embryology
in various classes of mammals (Bischoff, 1845, 1852, 1854;
Bonnet, 1884, 1891; Caldwell, 1887; Hartman, 1916, 1919;
Heape, 1883, 1886; Hensen, 1876; Hill, 1910, 1918; Hill and
Tribe, 1924; Huber, 1915; Hubrecht, 1912; Jenkinson, 1900,
1913; Keibel, 1888, 1894, 1899, 1901, 1902; Lams and
Doorme, 1908; Lams, 1910, 1913, 1924; Melissinos, 1907;
Minot, 1889; van Oordt, 1921; Reichert, 1861; Rein, 1883;
Robinson, 1892; Sakurai, 1906; Selenka, 1883, 1884, 1887;
Sobotta, 1893, 1895; Tafani, 1889; Van Beneden, 1875,
1880, 1899, 1911, 1912; Van Beneden and Julin, 1880; Weil,
1873; Wilson and Hill, 1907). The hving egg was neglected
presumably because no technique was developed for pre-
serving it intact in vitro long enough for any extensive
experimentation to be performed. Nor did the possibility
of experimental manipulation of ova in vivo receive more
than passing attention (see Grusdew, 1896; Novak and
Eisinger, 1923).
Since the pubhcation of Stockard and Papanicolou's (1917)
and Long and Evans' (1922) exhaustive accounts of the
oestrus cycle of the guinea pig and rat respectively, a new
era in the study of sexual physiology has been initiated.
Enormous strides have been made in the discovery and
purification of the hormones regulating the activities of the
genital tracts of mammals. The ovarian control of the various
phases of the sex cycles in the female has received exhaustive
attention, and the control of gonad function by the anterior
pituitary has been investigated in detail. Despite the enor-
mous accumulation of data on the endocrine regulation of
the ovarian and oviduct environment of ova, the ova them-
INTRODUCTION 3
selves have received relatively little attention. The study
of the hormonal control of ovarian function has centered
upon the relation of hormone activity to the development of
follicle and corpus luteum. The ovary has been largely
considered as a sort of diphasic machine geared for hormone
production by certain specialized follicle components. Its
primary function as a producer of gametes has been rela-
tively neglected. The endocrine control of the proliferative,
secretory and contractile activities of the oviducts them-
selves is known in detail, and it is tacitly recognized that
all these activities have as their end and aim the nutrition
and protection of the developing egg. Yet the exact nature
of the dependence of the o\aim upon these activities is still
problematical. We are now provided with the sort of
knowledge that should certainly make profitable in vivo
experimentation with eggs.
Brachet (1912, 1913) did indeed take advantage of the
development of a tissue culture technique in order to in-
vestigate a specific stage of development in rabbit ova.
But neither the availability of the technique nor Brachet 's
suggestive discourse led to any acti\^e investigation until.
1929 when Lewis and Gregory published their account of
the cinematography of rabbit ova developing in culture.
Since then a number of workers associated with Lewis
(Gregory, 1930; Squier, 1932; Lewis and Hartman, 1933;
Lewis and Wright, 1935) have conducted a fairly intensive
examination of living ova, chiefly with the object of cul-
turing fertilized eggs. In addition to these investigations
and similar work undertaken by Nicholas and his coworkers
(Nicholas and Rudnick, 1933, 1934; Defrise, 1933), the
physiological properties of developing ova have been ex-
amined from quite different angles. So there exists a meas-
urable body of work of recent origin which is properly
experimental. Wherever possible the factual data of this
work have been presented in the hope that these, speaking
for themselves, may stand side by side with any interpreta-
tion herein presented.
4 THE EGGS OF MAMMALS
It is the earnest belief of the writer that these experi-
mental inquiries represent a small fraction of the work
that should and will be done. The enormous variety and
richness of mammalian material that is available and un-
tapped should provide an extraordinary temptation to ex-
ploitation now that a beginning has been made in the de-
velopment of technical facilities for the manipulation of
this material. I emphasize that only a beginning has been
made. This book is a beginning.
CHAPTER II
THE ORIGIN OF THE DEFINITIVE OVA
A long-lived controversy concerns itself with the origin of
the definitive germ cells. Do they arise de novo from somatic
tissue in the sexually mature adult, or are they segregated
as primordial precursors early in embryogeny? Weismann's
theoretical considerations (1883, 1904, also Nussbaum, 1880)
on the continuity of the germplasm led initially to the active
investigation of this problem. In the light of modern the-
oretical genetics the strict interpretation of the Weismannian
dogmata is probably no longer necessary. For, since the
data of genetics indicate that every normal nucleus in the
organism contains the full complement of genes and that
somatic segregation of genes is a rare and exceptional phe-
nomenon, it is no longer necessary to postulate the trans-
mission of a special, unimpaired germ tissue. The problem
of the origin of the germ cells thus properly becomes one
concerned with the dynamics of embryonic differentiation
and peculiarly one of regeneration. In fact most of the
recent experimental approaches have been concerned with
the probability of the regeneration of germ cells from somatic
tissues. Able reviews of the general problem are contained
in the paper of Heys (1931) and the monograph of Harms
(1926).
Since we are concerned specifically with the origin of the
definitive ova of mammals the question that we may set is
concerned less with general theory and more with pertinent
fact. We want to know what processes are responsible for
the emergence in the ovary of the functional eggs.
We may at once distinguish two types of investigation.
The first, essentially descriptive, is concerned with the de-
velopment of the ovary and its germ cells from early em-
5
6 THE EGGS OF MAMMALS
bryonic life through sexual maturity. The second is con-
cerned with varying the conditions of ovarian growth by
experimental means and deducing from the derived data
the nature of the factors concerned in the production of
functional eggs. We shall assume that these two types of
observations are distinct, and consider them separately as:
(1) the morphogenesis of egg cells and (2) the experimental
investigation of the growth of egg cells.
The Morphogenesis of Egg Cells
Thanks to the Weismannian controversy we have avail-
able a fairly detailed description of oogenesis in embryonic
life. It is unnecessary here to enter into a detailed descrip-
tion of the embryogeny of the mammalian ovary (see
Jenkinson, 1913, de Winiwarter, 1901, de Winiwarter et
Sainmont, 1909, Brambell, 1927 and esp. 1930). Our inter-
est lies in the so-called ^'primordial" germ cells of the em-
bryo, since it is to these cells that a number of observers
trace the origin of the definitive ova.
The general opinion seems to be that large wandering
cells originate from the entoderm of the gut before or at
the time of the formation of the genital ridges (Nussbaum,
1880; Fuss, 1911, 1913). These primordial germ cells migrate
to the gonad site and enter the genital ridges. The ridges
are first seen as thickenings of the peritoneal epithelium
between the base of the mesentery and the Wolffian duct
on the ventral side of the developing mesonephros. The
thickened peritoneal epithelium becomes the germinal epi-
thelium and the primordial germ cells complete their migra-
tion when they become arranged beneath this epithelium
which then proliferates medullary tissue into the germ cells.
The underlying mesenchyme forms connective tissue trabec-
ulae in the medulla and also the primitive tunica albuginea
which separates the medulla from the germinal epithelium.
There are among investigators various opinions about the
role of the primordial germ cells. A number maintain that
these are the only germ cell precursors. The increase in
THE ORIGIN OF THE DEFINITIVE OVA 7
number of these cells is by mitosis only, and no new cells
are recruited from somatic tissue. This view is set forth at
some length by Hegner (1914, also Vanneman, 1917). It
leads naturally to the conclusion long maintained as a
biological truism that by the end of embryonic life or shortly
thereafter the complete quota of future eggs is attained
(c/. Waldeyer, 1870 and 1906; Felix, 1912 and Pearl and
Schoppe, 1921). The calculations of Aschner (1914) indi-
cating the presence of some 400,000 ova in the human ovary
at birth furnishes an apparent statistical substantiation.
Furthermore, meiotic phenomena are observable in these
primordial germ cells during embryonic and prepubertal
life (Cowperthwaite, 1925) but not thereafter, and the as-
sumption is made that typical meiosis is necessary for the
formation of definitive ova.
This conception of a large early store of future ova is
scarcely controverted by a second group of investigators
who admit the primordial germ cells as precursors of the
future ova, but who claim that additional egg cells are
supplied by proliferations from the germinal epithelium.
Brambell (1927) in a careful study of the developing gonads
of the mouse finds that the primordial germ cells persist
throughout embryonic life and undergo maturation stages,
but declares that additional cells from the germinal epi-
thelium must be responsible for the large increase of cortical
cells found in the gonad before the formation of the tunica
albuginea in ten and twelve day embryos.
Perhaps the largest group of observers consists of those
who also consider post-pubertal production of new egg cells
non-existent or negUgible but who find that the primordial
germ cells degenerate and are replaced by secondary pro-
liferations during embryonic or prepubertal life. Thus
Rubaschkin (1908, 1910, 1912) decided that the large dif-
ferentially staining primordial germ cells with their prom-
inent attraction spheres degenerate in the early guinea pig
embryo and are replaced by two successive proliferations
from the germinal epithelium. De Winiwarter and Sainmont
8 THE EGGS OF MAMMALS
(1909) describe a degeneration of the primordial germ
cells in the cat ovary and their replacement by ingrowths
from the germinal epithelium from three and one-half to
four months after birth {cf. Kingsbury, 1913 and 1914a;
Foulis, 1876 and Balfour, 1878). De Winiwarter (1910)
observed the same phenomena in human ovaries. In the
rat embryos Firket (1920) observed a secondary proliferation
following degeneration of the first generation of germ cells.
Kingery (1917) in a detailed study of oogenesis in the mouse
found that the definitive oocyte arose from secondary pro-
liferation begun at three to four days before birth and last-
ing until thirty-five to forty days post partum. He found
no evidence for oogenesis after puberty. In the rabbit
Buhler (1894) also found only prepubertal ovogenesis.
Simkins (1923 and 1928) questions the vahdity of the
term primordial germ cells, going so far as to state that in
the human embryo they are not large wandering cells at
all but large liquefied areas surrounding degenerating nuclei.
He attributes complete autonomy to the genital ridge.
Kohno (1925) recognizes primordial germ cells in the hu-
man embryo but declares their origin is in lateral plates of
the mesoderm whence they reach the gonad via the gut
epithelium and mesentery. Hargitt (1925) also denies the
peritoneal origin of the germ cells in rat embryos declaring
that large differentially staining cells are found throughout
the embryo in the epithelium, mesoderm, ectoderm, gut
entoderm and extra embryonic tissues. The disappearance
of these cells he attributes to division, not to migration
into the genital ridge.
A number of more recent investigators have observed a
more or less continuous proliferation of ova from the ger-
minal epithelium throughout life. The chief modern protag-
onists of this view are Robinson (1918), Arai (1920a and 6),
Allen (1923), Papanicolou (1925), Butcher (1927), Swezy
(1929a, 1933a and h) and Evans and Swezy (1931). Their
histological studies are essentially confirmations of earlier
observations on post natal ovaries (Pfluger, 1863 — cat;
THE ORIGIN OF THE DEFINITIVE OVA
Schron, 1863 — cat and rabbit; Koster, 1868 — man; Slawin-
sky, 1873 — man ; Wagener, 1879 — dog; Van Beneden, 1880 —
bat ; Harz, 1883 — mouse, guinea pig, cat; Lange, 1896 — mouse;
Coert, 1898 — rabbit and cat; Amann, 1899 — man; Palladino,
1894, 1898— man, bear, dog; Lane-Claypon, 1905, 1907—
rabbit; Fellner, 1909 — man) save that the work of Allen
and those who follow takes advantage of recent discoveries
of the nature of the oestrus
cycle, and presents observations
made upon ovaries taken at def-
inite times during the cycle.
Since the embryogenesis of the
primordial germ cells and the
germinal epithelium are separate loo —
and distinct it follows from the
findings of these observers that
the definitive ova of adult hfe
do not arise from the primordial
germ cells at all. Most of the
earlier workers observed evi-
dences of growth and thickening Fig. l. The frequency of mi-
of the germinal epithelium or r^^rttiaTa'cf AUe"!
even extensions of germinal epi- 1923. Open circles indicate com-
thelium into the ovarian cortex. ^^,f ^^^^ on semi-spayed mice.
Halt circles mdicate normal un-
In some cases these signs of operated controls. Abscissae are
activity were associated with stages of oestrus cycle; l, early pro
the period of heat.
Allen (1923) whose investiga-
tions are perhaps pioneer to
the most recent developments mitoses per mouse. (From the
distinguished four stages in the ^^nerican Journal of Anatomy.)
behavior of the germinal epithelium of the adult mouse
during the oestrus cycle. The first, characterized by ex-
tensive mitotic activity occurs just before and during oestrus
(see Figure 1). The second is marked by a fairly abrupt
decrease in mitosis frequency, and a position of the daughter
epithelial cells one cell layer below the germinal epithehum
oestrus; 2, late prooestrus; 3, pro-
oestrus to oestrus; 4, early oestrus;
5, oestrus; 6, early metoestrus;
7, metoestrus; 8, dioestrus. Or-
dinates are average number of
10
THE EGGS OF MAMMALS
due to the plane of cell division (Figure 2). In the third
stage the daughter cells extend two cell layers below the
epithelium. And by the fourth stage, occurring during
dioestrus, several hundred young ova surrounded by a few
folUcle cells are found just beneath the epithelium (Figure 3).
vm~'
Fig. 2. A late anaphase in the germinal epithelium of the mouse.
The plane of division is nearly parallel to the surface of the ovary.
(From the American Journal of Anatomy.)
According to Allen the tunica albuginea forms ^'from con-
nective tissue ingrowth during the absence of ovogenetic
proliferation of the germinal epithelium." Allen notes a
relatively intact tunica in animals that have had a long
period of dioestrus and also a complete or an almost complete
absence of young follicles.
Cowperthwaite (1925) has criticized Allen's data On the
grounds that he gives no demonstration of the presence of
meiosis in these presumable new ova. Typical meiotic
phenomena in adult ovaries have, in fact, rarely been ob-
served. De Winiwarter (1920) noted oocyte formation in
the region of the hilum in ovaries of cats shortly after puberty
but no such process in the remaining tissue, and Gerard
THE ORIGIN OF THE DEFINITIVE OVA
11
(1920) observed typical meiotic prophases in nests of young
oocytes in the adult ovaries of Galago. On the basis of
these observations and the presence of typical oocytes in
certain undescribed adult ovaries of Loris (material of
Prof. J. P. Hill and Dr. A. Subba Ran), Brambell (1930)
inclines to the belief that these primate oocytes derive from
primitive oogonia, not the germinal epithelium.
Fig. 3. A stage 4 ovum (see text) in the mouse. Note
complete layer of follicle cells. (From the American Journal
of Anato7ny.)
In rodents, however, such typical meiotic prophases have
never been described. Here the observations of Swezy
(1929a) and also of Evans and Swezy (1931), are very much
to the point and apparently resolve the mystery. Swezy
found the classical meiotic stages in the oocytes of rat
embryos and young rats up to five days post partum (Plate I,
Figs. 1-5), but she noted definite degeneration of all these
ova by the loth day post partum. By the 10th day
definitely atypical synizesis and pachytene stages occur
(Plate I, Figs. 6-13) and in 15 day old rats (Plate I, Figs. 14-
16) synizesis stages are rare or missing, the pachytene mod-
ified to a chromatin aggregation much less sharp than in
typical stages, and the diplonema chromosomes also less
distinct. On twenty day old rats (Plate II, Figs. 17-22)
nuclear growth of oocytes involves essentially similarly mod-
ifications, and in the adult the new ova derived from the ger-
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
f-'^fl:^ '#^ fT,« t->"-%
^
Fig. 7
Fig. 8
\ \
\ '^'
\ A 1
Fig. 9
,5 '"^^
^^
Fig. 10
Fig. 11
Fig. 12
Fig. 13
-.. r
1^
Fig. 14
Fig. 15
Fig. 16
Plate I. (From the Journal of Morphologij)
Figs. 1-5. Nuclei of ova from ovary of rat 5 days post partum. 1, Deutobroch
nucleus in germinal epithelium. 2, Leptotene nucleus. 3, Synizesis. 4, Pachynema.
5, Diplonema.
Figs. 6-9. Nuclei of ova from ovary of rat 8 days post partum. 6, Deutobroch
nucleus. 7, Synizesis. 8, Stage following 7, evidently modified pachynema. 9, Dip-
lonema.
Figs. 10-13. Nuclei of ova from ovary of rat 10 days post partum. 10, Deutobroch
nucleus. 11, Synizesis. 12, Modified pachynema. 13, Diplonema.
Figs. 14-16. Nuclei of ova from ovary of rat 15 days post partum. 14, Deutobroch
nucleus. 15, Modified pachynema. 16, Masses of chromatin changing into loose
threads.
12
'f^'-i-yl.
^:, -'^ :-/ ^"
, V»'
Fig. 17
Fig. 18
Fig. 19
Fig. 20
Fig. 23
Fig. 24
Plate II. (From the Journal of Morphology)
Figs. 17-22. Nuclei of ova from ovary of rat 20 days post partum. 17, Deuto-
broch nucleus. 18, Beginning of the formation of clumps shown in next figure.
19, Modified pachynema. 20, Later stage showing characters of diplonema. 21, Nu-
cleus toward the end of the growth period. 22, Final stage in twenty-day rat.
Fig. 23. Nucleus in mature follicle from adult rat. Fig. 24. Nucleus from ripe
follicle from adult rat.
13
14 THE EGGS OF MAMMALS
minal epithelium contain mature nuclei (Plate II, Figs. 23-
24) in which the modification presaged in the younger
animals attains culmination. These definitive ova show then
a modified type of meiosis which involves essentially the dis-
appearance of leptotene and synizesis, and the formation
of an atypical pachynema and diplonema. Evans and
Swezy (1931) obtained confirmation of these findings in the
guinea pig, cat, dog, monkey and man. They point out
that instead of being long-lived, the egg cells of mammals
are subject to heavy mortality and exhibit a very short
life cycle, correlated apparently with the length of the normal
ovarian rhythm. In those animals in which the oestrus and
ovarian cycles coincide {e.g., rat, mouse, guinea pig) the
length of the oestrus cycle is a measure of the lifetime of the
ovum in the ovary.
These rather straightforward histological findings seem
to indicate, on the whole, that the definitive ova originate
from the germinal epithelium. All our recent knowledge of
the rhythmic activity of the ovary with its periodic produc-
tion of large numbers of young ova (Allen, Kountz and
Francis, 1925) militates against the assumption of a single
large initial store of ova gradually being exhausted through-
out sexual maturity.
The Experimental Investigation of the Growth of
Egg Cells
Any attempt to analyze the experimental data pertinent
to the problem of the origin of the definitive ova encounters
two difficulties. First of all many of the experiments are
concerned with the simple Weismannian problem and ignore
certain now obvious endocrinological implications. And
secondly, the difficulty of experimental treatment of mam-
malian embryos makes for a hiatus in our knowledge that
can only be bridged by indirect deduction.
The information that we do have at hand is derived from
experiments concerned with the effects resulting from (1) bi-
lateral ovariectomy, (2) partial ovariectomy, (3) ovarian
THE ORIGIN OF THE DEFINITIVE OVA 15
transplantation, (4) the irradiation of ovaries with x-rays,
(5) hypophysectomy, (6) the injection of gonad-stimulating
hormones and (7) the transplantation of embryonic gonad
rudiments.
Bilateral ovariectomy has been extensively employed in
order to determine whether ovarian tissue and eggs can be
derived from somatic cells. It is a common experience that
ovariectomized animals apparently regenerate ovarian tissue
some time after the operation. Thus Davenport (1925)
observed as many as 64 per cent of bilaterally ovariecto-
mized mice with apparently functional ovarian tissue ap-
pearing within a few weeks to several months after the
ovariectomy. Such data may be explained as due either to:
(1) regeneration of germinal tissue de novo from somatic
cells or (2) the presence of accessory gonadal tissue distinct
from the ovary and not removed during the operation or
(3) the incomplete removal of ovarian tissue so that frag-
ments remaining hypertrophy and attain dimensions suf-
ficient to permit the manifestation of ovarian function. If
the first alternative is accepted then it follows that neither
germinal epithelium nor, presumably, primordial germ cells
are necessary for the production of ova. The two latter
alternatives exclude the first but scarcely affect the problem
of origin via germinal epithelium or primordial germ cell
though careful observation of the process of hypertrophy
may yield pertinent data. Even if the first alternative is
acceptable and may thus very well settle the ghost of germ-
plasm continuity, it does not necessarily inform us about
the normal process of egg production.
In rodents accessory gonadal tissue is rarely, if ever,
present. On the other hand, it is known that fragments of
ovarian tissue, remaining after incomplete extirpation of
the ovaries, will hypertrophy to such a remarkable degree
that a completely normal ovary will be reestablished from
which fertilizable ova are liberated (c/. Haterius, 1928; and
Pincus, 1931). Furthermore it is quite possible to fail to
extirpate small fragments of the irregularly lobed encap-
16 THE EGGS OF MAMMALS
sulated rat and mouse ovaries, or even after careful excision
to drop very small crushed fragments. A number of in-
vestigators have therefore repeated Davenport's experiments
using extreme operative precautions, in some instances going
to the trouble of making serial sections of the extirpated
ovaries in order to be certain of the completeness of removal.
In practically every instance the per cent of animals
showing return of oestrus symptoms or of detectable ovarian
^X_J
Fig. 4. Section through ovary of young
rat showing small, compact ovary. YF,
young foUicle; C, ovarian capsule. LL,
line of excision. (From the Quarterly Re-
view of Biology.)
tissue has been much below that reported by Davenport.
Fallot (1928) found return of vaginal cornification in three
out of twelve ovariectomized rats within six to six and one-
half months after operation, and ovarian tissue was found
in two of these. Parkes, Fielding and Brambell (1927)
detected oestrus symptoms after operation in eleven out of
one hundred and twenty-one mice, identifying ovarian tis-
sue in eight of these eleven. Haterius (1928) also found
apparent regeneration in 10 per cent of the mice he ovari-
ectomized, and attributed the regeneration to incomplete
extirpation. Pencharz (1929) reported return of oestrus in
only three of 118 ovariectomized rats and mice, and demon-
strated by serial sections of the ovarian region that incom-
plete removal had been made in the case of these three.
Heys (1929 and 1931), in an extremely careful analysis of
THE ORIGIN OF THE DEFINITIVE OVA
17
a series of double ovariectomies in the rat, has demonstrated
the presumable source of regenerated tissue in animals with
apparently completely extirpated ovaries. In an initial
series of 105 double ovariectomies she found germ cells at
the ovarian site in eight cases, and observed that all eight
YF
-».FA
Fig. 5. Section through the ovary of mature
rat showing the iobed condition. YF, young fol-
licle; F, follicle; FA, fatty tissue. (From the
Quarterly Review of Biology.)
occurred in the sixty animals over forty days of age. She
noted that in females under forty days of age the ovary is
relatively smooth and compact and not very heavily em-
bedded in fat (Figure 4), whereas in older animals the ovary
is Iobed and surrounded by a la]:ger amount of fat (Figure 5).
She accordingly ovariectomized a second set of animals con-
sisting of eighty-five females under forty days of age and
twenty-three older females. Three of the older animals re-
generated germ cells but none of the younger ones did. In
several of the positive cases serial sectioning of the removed
ovaries gave no detectable indication of lost fragments, but
Heys believes that certain narrowly constricted lobes of
18 THE EGGS OF MAMMALS
ovarian tissue might very well be lost and the loss not
noticed upon serial sectioning (see Figure 5). Heys' results
can scarcely be due to chance alone, the difference in regen-
eration incidence between the young and older rats being
3.43 times the standard error of the difference, i.e., the odds
are over 3000 to 1 against this being a chance difference.
It is clear, therefore, that regeneration of ovogenetic tissue
from somatic tissue is improbable in mammals. And cer-
tainly the definitive ova are normally not recruited from
somatic cells. We must turn to other experimental pro-
cedures to obtain some insight into the processes that lead
to the birth of ova in normal functional ovaries.
The simple observation that unilateral ovariectomy or
incomplete total ovariectomy leads to a compensatory hyper-
trophy of the remaining tissue has led to a long series of
researches which, often incidentally, form the basis for our
modern knowledge of the elements of ovarian dynamics.
The fact that such hypertrophy occurs was originally estab-
lished both clinically (Robertson, 1890; Gordon, 1896; Sut-
ton, 1896; Morris, 1901; Doran, 1902; Kynoch, 1902; and
Meredith, 1904) and experimentally (Kanel, 1901; Bond,
1906; Carmichael and Marshall, 1908). An almost exact
doubling of weight in the remaining ovary of unilaterally
ovariectomized rats has been reported by Stotsenburg (1913)
and Hatai (1913, 1915) and the number of eggs shed is
demonstrably equal to the number normally produced by
two ovaries (see Lipschiitz, 1924; Hanson and Boone, 1926;
Crew, 1927; and Slonaker, 1927). In the opossum Hartman
(1925) has reported a tripling of the weight of the remaining
ovary and a similar threefold increase in the number of eggs
shed. In the rabbit (Asdell, 1924; Hammond, 1925; Lip-
schiitz, 1928) and the cat (Lipschiitz and Voss, 1925) a
single remaining ovary or even small ovarian fragments
produce the typical adult number of ripe follicles and eggs,
but an exact compensatory hypertrophy of ovarian tissue
is not so evident. Emery (1931) in a large series of uni-
laterally ovariectomized rats found not a doubling in weight.
THE ORIGIN OF THE DEFINITIVE OVA 19
but a one and one-half times compensatory hypertrophy
when careful comparison with a control series was made.
It is significant that in Emery's material about 50 per cent
of the rats were found at autopsy to have large ovarian
cysts. Similar cystic formations were observed in about
half of the semi-spayed females in Wang and Guttmacher's
(1927) series, and Wilhams (1909) reports that such cysts
are commonly found in ovarian fragments left after incom-
plete ovariectomy.
Arai (19205) found definitely that the compensatory hyper-
trophy in the rat is due exclusively to an increase in the
number of large follicles and corpora lutea. Semi-spaying
before puberty when the formation of corpora lutea does not
normally occur led to a 40 per cent increase in ovarian weight,
whereas semi-spaying after puberty led to a 100 per cent
increase. Furthermore, by careful counts he established
that the total number of follicles in the ovary does not in-
crease after semi-spaying. In this Arai was confirmed by
Alien (1923) who found that in semi-spayed mice the num-
ber of ova differentiating from the germinal epithelium
during stages 2 and 3 (vide supra) was scarcely larger than,
normal whereas the average number of mature ova formed
was normal. The implication from these studies is that the
germinal epithelium produces a large more or less constant
number of young ova, that some extra-gonadal factor is
responsible for the ripening and maturation of a Hmited
number of follicles, and that the maturing crop of ova are
chiefly involved in the compensatory hypertrophy. It is
now well established that an enormous atresia of young
follicles occurs during the course of a single oestrus cycle.
Thus in swine 14 per cent of the visible follicles less than
3 mm. in diameter become mature (Allen, Kountz and
Francis, 1925) and in the rat of the ova less than 20 fx in
diameter only 0.8 per cent attain a diameter greater than
60 M (Arai, 1920a). This extensive destruction of young ova
and follicles is particularly striking in the dog and cat
(Evans and Swezy, 1931) where all the new eggs (except
20 THE EGGS OF MAMMALS
those ovulated) formed in the metoestrum and anoestrum
preceding ovulation are completely degenerated by the time
of ovulation.
That the germinal epithelium is the source of new ova
formed in hypertrophying ovarian tissue is demonstrated
by the behavior of transplanted ovarian tissue. Among
those who have observed the histological development in
such tissue only Marshall and Jolly (1907, 1908) report
complete disappearance of germinal epithelium with reten-
tion of function. Lipschlitz (1928) notes a decrease in the
number of primary oocytes in small fragments of rabbit
ovaries in incomplete ovariectomy when comparison is made
with similar sized fragments isolated from the ovaries in
unovariectomized controls. But it is notable that his proto-
cols describe a partially preserved or ''flattened" (degenerat-
ing?) germinal epithelium in the experimental group whereas
the germinal epithelium in the control fragments is appar-
ently much better preserved. Tamura (1926) examining a
series of ovarian transplants made onto the kidneys of male
mice found the presence of primary follicles and many
young ova associated with an actively mitotic germinal
epithelium. Where the degree of activity of the germinal
epithelium is less and more varied, small and medium sized
or various sized folUcles are present. Apparently the activ-
ity of the germinal epithelium is largely conditioned by the
pressure of overlying connective tissue growths since its
activity is greatest at free surfaces. Nonetheless, Tamura
claims a rhythmical proliferation of ova from the germinal
epithelium, but assigns a length of ten days to the ovogenetic
cycle which is twice the length of the normal five-day oestrus
cycle. Schultz (1900) and Voss (1925) also observed the
persistence of functional germinal epithelium in their series
of transplantations, but offer no such detailed an analysis as
Tamura. Butcher (1932) has examined the nature of ovo-
genesis in ligated ovaries and in autotransplantations of
ovarian fragments and observed that the development of
young ova is definitely associated with the activity of the
THE ORIGIN OF THE DEFINITIVE OVA 21
germinal epithelium. Furthermore, in the hgated ovaries
the follicles become necrotic and new ova are proliferated
from the germinal epithelium which is relatively unimpaired.
Athias (1920) has described proliferation of new ova from
the germinal epithelium of transplanted guinea pig ovaries.
No attempt has been made to make a quantitative study of
the relation between the number of new ova formed and
the amount of functional germinal epithelium in trans-
planted or fragmented ovarian tissue, but it seems evident
that the formation of new ova in such tissue occurs in the
germinal epitheUum. Thus in Tamura's material the few
cases of degenerated transplants were marked by a complete
absence of germinal epithelium.
It is possible, however, to preserve an intact germinal
epithelium with total disappearance of follicles in x-rayed
ovaries (Parkes, 1926, 1927a, b and c; Brambell, Parkes and
Fielding, 1927a and h). Parkes and his coworkers have
described in some detail the replacement of degenerated
folUcular tissue by cellular proliferations from the germinal
epithelium in the irradiated ovaries of mice. These pro-
liferations never give rise to ova, however, though the ovaries
seem to retain their hormone-producing capacities as evi-
denced by the continuance of oestrus cycles of normal length
in the irradiated animals. In the ferret (Parkes, Rowlands
and Brambell, 1932) x-ray sterilization is also marked by an
obliteration of the follicles and oestrin secretion, whereas
in guinea pig ovaries (Genther, 1931, 1934) a transformation
to luteal tissue usually occurs with only occasional follicle
formation. Brambell (1930) inclines to the belief that the
destruction of primordial ova is responsible for the lack of
ovogenesis, but it is equally likely that the x-rays affect
differentially the ovogenetic and hormone-producing capac-
ities of ovarian tissue. It is notable therefore that the pro-
liferation of new tissue from the germinal epithelium in
x-rayed mice resembles the production of anovular follicles.
Hill and Parkes (1931) have attempted to induce germ cell
formation in mice with irradiated ovaries by means of in-
22
THE EGGS OF MAMMALS
jections of pituitary and pregnancy urine extracts, but no
ova were ever produced in the injected animals.
That the early stages of ovogenesis in adult ovaries are
scarcely under the control of pituitary hormones is abun-
dantly evident from observations made upon the ovaries
of hypoph3^sectomized animals. Smith (1930) noted that in
completedly hypophysectomized rats no new large folUcles
or corpora lutea develop, but the proliferation of young
follicles goes on unimpaired for many months after hypoph-
ysectomy. Swezy (19336) has presented quantitative meas-
ures of the rate of ovogenesis in hypophysectomized rats,
and her data indicate that a larger number of young ova
may be produced in hypophysectomized females than in
normal non-pregnant animals. In Table I is presented a
suEMnary of her findings.
TABLE I
Numbers of Ova, Follicles axd Corpora Lutea in a Single Ovary of
THE Rat during the Oestrus Cycle, Pregnancy and Pseudopreg-
NANCY, and after Hypophysectomy AND THYROIDECTOMY. (From Swezy,
19336)
Day of
Average
Stage
Num-
ber
OF
Rats
Cycle
(or Days
AFTER
Oper-
ation)
Age,
Days
Number
of Ova
AND
Primary
Follicles
Average
Number
of
Larger
Follicles
Average
Number
OF
Corpora
Lutea
Total
Oestrus cycle
5
2nd(l),
4th (4)
206-208
1809
171
27
2007
Pregnant and
pseudopreg-
nant
10
5 to 22
98-224
3857
311
16
4184
Hypoph3'sec-
tomized
8
12 to 90
95-202
4164
—
20*
4184
Thyroidec-
tomized
3
36 to 42
403
1371
193
15
1579
* Persisting old corpora.
Swezy concluded from these data that there is a basic
rate of ovogenesis which is observed in hypophysectomized
animals. That the increased number of ova in hypophysec-
tomized animals is due to an increased rate of production
and not merely to accumulation is proven by the absence
of any unusual number of degenerated ova. This rate is
THE ORIGIN OF THE DEFINITIVE OVA
23
decreased when the hypophysis is secreting active maturity
hormone as in non-pregnant females. The maturity hormone
is concerned with the ripening of large follicles, ovulation
and corpus luteum formation. During pregnancy and pseu-
dopregnancy maturity hormone is secreted only in sub-
threshold amount, as evidenced by cyclic ovarian changes
OVA OF LARGER SIZES
\
\
A -20 4
B -40-f
C-OV
0"
400 -
\
ER 60 "
200 -
iW-
/•^.
— X-
-^
— xB
100 -
.....X.— J
*^
-
NUMBER OF OV^
\ (TOTA
L)
:f^z-:
— ^C
^,
V
_ ^
• t _
—
—
100
200
300
700
800
900 1.000
400 500 600
AGE - DAYS
Fig. 6. Showing the total number of ova as well as the number of ova of
different sizes in the albino rat at different ages (condensed). (From the
American Journal of Anatomy.)
in the ovary during pregnancy (Swezy and Evans, 1930),
so that the hypophysectomized level is attained. During
the normal non-pregnant ovogenetic cycle that portion
marked by the presence in the ovary of ripe follicles and
fresh corpora lutea is always associated with a minimum of
small, newly formed ova. The pituitary secretions, then,
are concerned with promotion of o\ailation and luteinization
and presumably inhibit ovogenesis to a certain extent. The
factor controlling ovogenesis is unknown although the effects
of thyroidectomy indicate that the thyroid may promote
ovogenesis to a certain extent. It should be pointed out,
however, that the thyroidectomized rats were much the eld-
est of the lot and Arai (1920a) has demonstrated a small
24 THE EGGS OF MAMMALS
decline of ovogenesis with age in adult females (see Fig-
ure 6) .
The experiments of Engle (1928) demonstrate adequately
that pituitary secretions are responsible for the later stages
of maturation. He injected anterior lobe tissue into normal
and semi-spayed rats and found that the per cent of hyper-
trophy due to pituitary stimulation was approximately equal
in the two groups of animals. We have already noted that
in compensatory hypertrophy the increased ovarian weight
is due to the doubling of large follicle and corpus luteum
number, the number of primary follicles being the same in
a single ovary whether the second ovary is present or
not.
Swezy (19336) also determined the effect of various pitu-
itary hormone preparations upon ovogenesis in adult and
immature rats. Her data are collected and summarized in
Table II.
Immediate verification of the conclusions deduced from
Table I is found in the data derived from the injection of
rat hypophyses into adult and immature rats (columns [11,
[10] and [11]). Rat hypophyses are notably rich in gonad
stimulating hormones (Smith and Engle, 1927), and their
administration results in a decrease in the rate of ovogenesis,
and an increase in total ovarian tissue. The data on the
immature rats are particularly striking, for a few days of
pituitary administration results in a halving of the total
number of ova. Arai (1920a) found that the average total
number of ova in prepubertal rats was about 10,000 and
approximately 6000 in post-pubertal animals.
Beef hypophyses, on the other hand, are relatively poor
in maturity hormone and rich in growth hormone. Evans
and Simpson (1928) have demonstrated an antagonism be-
tween the growth and gonad-stimulating hormones of the
anterior pituitary. The increase in follicle number following
beef hypophysis administration (column 2) might then be
interpreted as a neutralization of the intrinsic maturity
hormone effect by the growth hormone of the beef pituitary.
TABLE II
The Number of Ova, Follicles, Cysts and Corpora Lute a in Sin-
gle Ovaries of Rats Subjected to Various Hormone Treat-
ments. (From Swezy, 19336)
Treatment
(1) Rat
hypoph-
ysis
(2) Beef
Vsc.c.
hypoph-
ysis
(3) Beef
Vs c.c.
hypoph-
ysis
plus rat
hypoph-
ysis
(4) Preg-
nancy
urine
(5) \U c.c.
theeUn
(6) 21-34 c.c.
follicular
fluid
(7) V4-I c.c.
growth
hormone
(8) V4-I c.c.
growth
hormone
(9) 0.5 c.c.
growth
hormone
(10) Control
(11) Rat hy-
pophysis
No.
OF
Rats
Age
OF
R.A.TS
(Days)
Days
OF .\D-
MINIS-
TRA-
TION
Ova
AND
Pri-
mor-
dial
Fol-
licles
Large
Fol-
licles
Corpora
Cysts
To-
tals
1661
5
153-182
9-20
1436
155
58*
12
4
153-168
9
4017
228
20
3
4268
1
154
9
1476
295
4
none
1813
2
172-174
10
3322
216
20
12
3570
6
181-190
18
3574
245
22
none
3841
6
183-254
10-14
2183
203
18
none
2404
6
255
35-97
4996
144
3
5143
2
255-408
60
and
394
2277
190
60
—
2527
2
1
139
and
141
24
9
1952
7225
300
31
—
2283
7225
5
24- 26
2- 8
3664
—
present
in some
3664
Weight
MGMS.
227=*
42
97
59
26
sub-
nor-
mal
(3)
and
hy-
pophy-
secto-
mized
types
76
ma-
turity
type
35
(mixed
type)
9.5
67t
* Varied with amount of hypophysis.
t Average of three.
25
26 THE EGGS OF MAMMALS
Simultaneous injection of beef and rat hypophysis tissue
results in inhibition of ovogenesis (column 3).
When, however, examination was made of the ovaries of
animals receiving injections of growth hormone extracts
various results were obtained. In six of the ten animals ob-
served (column 7) the expected result was obtained, namely
an inhibition of ovarian growth and a rise in the rate of
ovogenesis. Two animals (column 8) with normal, good
sized ovaries exhibited a normal rate of ovogenesis, and
two animals (column 9) with somewhat decreased ovarian
weight gave no indication of increased ovogenesis. Two
interpretations of these data are possible: (1) the growth
hormone preparations may in some instances have con-
tained sufficient maturity hormone to overcome the typical
growth hormone effect or (2) there may have occurred in
some of the injected animals a conversion of growth hormone
to maturity hormone (c/. Evans, Meyer and Simpson, 1932;
Evans et at., 1933). It should be pointed out that Reiss,
Selye and Balint (1931a, h) have obtained from the pituitary
extracts free of growth hormone which also inhibit the
action of maturity hormone. Swezy's extracts are not made
in a manner that would free her preparations of such ma-
terials. Obviously the use of highly purified extracts and
carefully timed injections should assist in resolving the
situation.
Pregnancy urine extracts (column 4) seem to increase
ovogenesis to some extent. It is known that pregnancy
urine is only partially effective as a maturity hormone
(Engle, 1929; Evans and Simpson, 1929).
Prolonged oestrin injection is known to reduce ovarian
growth (Doisy, Curtis and Collier, 1931; Leonard, Meyer
and Hisaw, 1931; Spencer, D'Amour and Gustavson, 1932;
Pincus and Werthessen, 1933), presumably by inhibiting
secretion of maturity hormone from the anterior pituitary
(Meyer, Leonard, Hisaw and Martin, 1932). One would
expect therefore that the data of columns 5 and 6 should
show an enhanced ovogenesis. It is interesting to note
THE ORIGIN OF THE DEFINITIVE OVA 27
that this seems to be the case when relatively light oestrin
doses are injected (column 5), but not with heavy doses
(column 6). The theelin-injected animals received about
6.25 r.u. per day, and while continuous vaginal cornification
resulted, an apparently normal cycle of uterine changes
occurred and the ovaries appeared relatively unimpaired.
It is possible that in the animals receiving light doses the
ovogenesis inhibiting capacity of maturity hormones was
impaired but not the follicle stimulating capacity. The
heavier dosages may have caused the hydropic degeneration
of the germinal epitheUum described by Doisy, Curtis and
Collier (1931) and so prevented maximum ovogenesis, al-
though Swezy makes no note of such degeneration. Swezy,
noting that normally during the oestrus cycle there is a
drop in the production of new ova at the period just suc-
ceeding the period of maximum oestrin production (e.^.,
ovulation), is inclined to attribute this drop (and therefore
the results in her oestrin-injected animals) to a factor other
than the ''suppression" of hormone secretion from the
pituitary.
Recently Hisaw and his collaborators have advanced an
explanation of the oestrus rhythm which involves a sep-
aration of the maturity principle of the pituitary into two
hormones (Fevold, Hisaw and Creep, 1934; Lane and Hisaw,
1934; Hisaw, Fevold, Foster and Hellbaum, 1934; and Lane,
1935). One hormone is follicle stimulating, the other lutein-
izing and a chemical separation of the two has been attained
(Fevold, Hisaw and Leonard, 1931 ; Fevold and Hisaw, 1934).
These investigators report an increase in the total number
of follicles in rat ovaries on administration of follicle stim-
ulating hormone to prepubertal rats but no increase when
luteinizing hormone is administered. Their count of ''total
follicles" includes only ova in definitely formed follicles.
Swezy (19336) attributes the ovogenesis inhibition to the
luteinizing hormone. It is possible, therefore, that in addi-
tion to the ovogenetic activity which is independent of the
hypophysis {e.g.^ the ovogenesis seen in hypophysectomized
28 THE EGGS OF MAMMALS
animals) a stimulation to ovogenesis may be engendered by
the follicle stimulating hormone. Hisaw and his collab-
orators find that corporin (the hormone of the corpus luteum)
exerts effects on the ovary like those of the follicle stimulat-
ing hormone while oestrin decreases the secretion of follicle
stimulating hormone and stimulates luteinizing hormone
production from the hypophysis. Pregnant and pseudo-
pregnant animals may therefore exhibit an increase in ovo-
genesis due to direct action of corporin from their corpora
lutea, whereas animals in oestrus and those receiving oestrin
injections show reduced ovogenesis perhaps because of the
action of the induced luteinizing hormone secretion.
It is obviously not possible to arrive at any final decision
concerning the factors governing ovogenesis until additional
pertinent data are available. The most concise summary
of the evidence indicates that ovogenesis occurs from the
germinal epithelium at a typical intrinsic rate which may
be reduced by the action of a hormone or hormones from
the anterior pituitary. But even this deduction requires
further verification in the form of careful quantitative esti-
mates of ovogenesis in its relation to atresia, and particularly
an inquiry into the nature of the atresia of young ova and
folhcles. We are completely unaware of the intimate nature
of the intrinsic proliferative capacity of the germinal epi-
thelium. How does it compare with the mitotic index of
tissues generally? Is it a self-perpetuating phenomenon in
the sense that the atresia of its products releases substances
stimulating cell division? We shall see for example that the
atresia of maturing follicles is often accompanied by the
formation of mitotic spindles and it is well known that
cytolized cell products (trephones) promote cell division.
An extraordinary variety of problems suggest themselves.
Patience and the formation of substantiated hypotheses will
result in their solution.
In summating the evidence relating to the normal ovo-
genetic processes in prepubertal and post-pubertal animals
little doubt remains that the definitive ova are proliferated
THE ORIGIN OF THE DEFINITIVE OVA 29
from the germinal epithelium. What then is the role of the
primordial germ cells of the embryo? Are they essential
structures or merely incidental? There are practically no
illuminating experimental data on the development of em-
bryonic gonads. The experimental manipulation of mamma-
lian embryos is dependent upon the elaboration of techniques
now in the process of initiation. Certain investigations
of gonadogenesis in amphibian and chick embryos offer
provocative suggestions, but their applicability to mammals
has yet to be proven.
In the chick a gonad or gonad-like organ may form free of
primordial germ cells. This can be demonstrated by removal
or destruction in three to nine somite embryos of the anterior
crescent in which the primordial germ cells originate. The
embryos nonetheless develop small gonad rudiments (Rea-
gan, 1916; Benoit, 1930). Willier (1932, 1933a and h) has
excised the germ cell crescent and transplanted the entire
blastoderm and found a sterile gonad developed in the
transplant. In the frog (Kuschakewitsch, 1910) sterile
gonads free of germ cells develop from the genital ridge
when delayed fertilization prevents germ cell migration,
Humphrey (1928), on the other hand, finds that in Ambly-
stoma gonads form in grafted tissue only when a sufficient
number of primordial germ cells are located beneath the
coelomic epithelium which gives rise to the germinal epi-
thelium. '
It is notable that in all instances gonads arising free of
primordial germ cells are sterile. Thus Domm (1929) found
in the fowl that if the large functional left ovary is removed
prior to the time of the disappearance of the germ cells from
the small rudimentary right gonad the latter develops into
a testis which produces sperm. If excision of the left ovary
is delayed until the time when the germ cells of the right
gonad are no longer present (the germ cells normally dis-
appear by the third week after hatching) a sterile testis
develops.
Willier (1933a and h) has demonstrated by means of
30 THE EGGS OF MAMMALS
chorio-allantoic grafts of the gonad-forming areas of chick
gonads that germ cells remaining outside the germinal ridge
area do not differentiate into oogonia or spermatogonia,
whereas those that become situated under the germinal
epithelium develop as typical sex cells. On the basis of
this and other evidence he agrees with Witschi (1929) that
the cortex {e.g., the cortical sex cords) of the gonad acts
upon the germ cells as a specific organizer of female sex
cells, and the medulla as organizer of spermatogenetic tissue.
In the free-martin of cattle, which is a female twin develop-
ing in utero under the influence of the hormones of its male
partner, a sterile testis-like organ develops. It is notable
that while typical male sex cords are present, germ cells
are absent (Chapin, 1917; Willier, 1921). Perhaps in the
case of the free-martin (as in the frogs with delayed ferti-
lization) a spermatogenetic tissue is not formed because
primordial germ cells do not reach the gonad.
If these data are generally applicable to manamals it would
seem that although ovogenesis takes place from the germinal
epithelium the formation of a functional ovary is dependent
upon the primordial germ cells. We have seen, in the case
of x-rayed ovaries, that an ovary with morphologically
normal germinal epithelium may be incapable of forming
ova. A necessary mechanism is lacking. It may be that the
primordial germ cells are the precursors to this mechanism
in normally developing ovaries.
The evidence from the free-martin and recent data on the
transplantation of embryonic gonad rudiments indicates
that, as in amphibia and birds, the development of an ovary
in embryogeny is dependent upon the formation of a cortex
in the developing gonad. Normally in ontogeny the gonads
of both sexes are morphologically indistinguishable for some
time. The genital ridge, as already noted, consists of ger-
minal epithelium overlying primordial germ cells. At about
the 10 mm. stage in both the pig (Allen, 1904) and cat
(Sainmont, 1905) and at the 12th day post coitum in the
mouse (Brambell, 1930) the germinal epithelium begins to
THE ORIGIN OF THE DEFINITIVE OVA 31
proliferate the primary sex cords from its inner surface.
During the formation of these cords (or nest of medullary
cells as in man [Felix, 1912]) the gonad is still morphologi-
cally indifferent. Morphological differentiation may be con-
sidered as initiated when these primary cords become iso-
lated in the medulla by the formation of the primitive tunica
albuginea under the germinal epithelium in the male gonad
and the proliferation of a second set of cortical sex cords
from the germinal epithelium in the female gonad. In the
embryonic ovary the medullary cords persist for some time
but are rarely found after birth; the cortical cords break up
to form primitive follicle cells surrounding the primordial
ova.
Buyse (1935) has transplanted rat gonads in the morpho-
logically indifferent stage onto the kidney of adult rats of
both sexes. Over 60 per cent of the transplants developed
as testes, 16 per cent as ovaries and the remainder were
bisexual gonads or gonads of undetermined sex. A small
percentage of the gonads classified as rudimentary testes
seemed to be transformed ovaries. It will be seen that if
these are included in the group of gonads other than testes
the normal sex ratio is approximated. Since the type of
gonad developed was not correlated with the sex of the
host Buyse concludes that adult sex hormones do not affect
sex differentiation. The differentiation was then dependent
on the history of the sex cords in the transplanted tissue.
Presumably the clear cut segregation of testes was due to
the presence of formed primary sex cords, e.g., the testis
organizers, whereas various types of zygotic ovaries were
obtained dependent on the probability of formation or
partial formation of the cortical sex cords.
CHAPTER III
THE GROWTH OF THE OVUM
We have seen that the production of ova from the germinal
epithehum may proceed in the absence of the hypophysis.
But does the formation of mature ova depend upon hypo-
physeal hormones? It is clear that ovulation and particularly
the number of follicles that liberate ova is dependent upon
hypophyseal hormones. Does this dependence involve
merely a maturation of the follicular apparatus or is the
actual growth of the ova also concerned? In fixed material
cells distinguishable as primary ova are in the mouse a
little less than 7 microns in maximum diameter (Pincus,
unpublished data), in the rat 8 microns (Aral, 1920a). They
eventually attain maximum diameters of 65 to 70 microns.
What are the factors governing the growth of these ova to
maximum size?
While direct measurements are unavailable it seems obvi-
ous that in hypophysectomized animals the ovum attains
the maximum size. Smith (1930) notes that the primary
follicles in hypophysectomized rats ^^continually are under-
going development, but invariably undergo atresia not later
than the stage of cavity formation." Swezy (1933) notes
the presence of a follicle having a diameter of 270 microns
in a rat ovary 90 days after hypophysectomy and mentions
foUicles with diameters of 200 microns. It is evident from
the figure in Selye's (1933) paper that foUicles with antra
occur in 43 day old rats hypophysectomized at 18 days of
age. In the dwarf mouse the largest follicles are about
200 microns in diameter and contain antra (Pincus, un-
published data).
Now it has been demonstrated (Brambell, 1928) that in
the mouse the diameter of the follicle when the ovum is
32
THE GROWTH OF THE OVUM 33
fully grown is 125 microns and in the rat (Parkes, 1931)
the maximum diameter of the ovum is attained when the
folhcle is 160 microns in diameter. Full growth of the
ovum, then, is attained just before the time of antrum
formation which begins in rats and mice in follicles having
diameters of about 200 microns. We may therefore deduce
that the ova of hypophysectomized animals attain the di-
mensions of the mature ova in o\ailating animals, and that
the growth of the ova (and early follicular growth) is inde-
pendent of the hypophysis.
This conclusion is supported by various independent lines
of evidence. Aral (1920a) found that ova over 60 ijl in diameter
appear in the ovaries of rats between the 15th and 20th days
of age. Engle (1931a) found pseudomaturation spindles,
which appear only in ova of full size, first evident in 16 day
old mice and no follicles more than 180 /z in diameter in
14 day old mice. Smith and Engle (1927) found that 10 day
old mice treated with gonad-stimulating pituitary implants
had to have daily implants for 5 days in order that full
ovarian response should be attained, whereas 17 day old
mice showed full response in 36 hours to 3 days. Corey
(1928) found practically no ovarian response to pituitary
extracts in rats until after the 15th day, and Selye and
Collip (1933) found no follicular maturation in 6 to 12 day
old rats treated with anterior pituitary-like hormone (see
also Zondek, 1931). In rabbits (Hammond and Marshall,
1925) the antrum develops later than the 10-1 1th week of
life. Hertz and Hisaw (1934) were able to obtain definite
follicular response to follicle-stimulating and luteinizing hor-
mones only in juvenile rabbits (12 to 13 weeks old), not in
infantile rabbits. Casida (1935) reports that pig ovaries show
definite response to pituitary hormones only when antrum-
containing follicles are present.
Nonetheless, fully potent pituitaries are present in 5 to
8 day old rats (Smith and Engle, 1927; Lipschutz, Kallas
and Paez, 1929) as judged by their effects in transplantation
to immature recipients. It would seem, then, that the at-
34
THE EGGS OF MAMMALS
tainment of a certain degree of follicle maturity and full
ovum size is necessary before activation of the pituitary
hormones can be attained in developing animals. It is to
H
O
o
o
p, 50
O
10
X
J«i. . X-rX— X-
_x__x_^x-
X X
V
r
X X
X X
x —
X
-/
/
'
/
1
1
1
1
1
100
500
600
2U0 300 4U0
DIAMETER OF FOLLICLE
Fig. 7. Showing the relation of ovum growth to folhcle growth. Data on the
mouse. (From Brambell, 1930, courtesy of The Macmillan Company.)
be remembered, however, that the release of substances
from the normal gland in vivo and the injection of excised
70-
>
o
o
g 40-
H
<
50
30
20
(b)
/ •:
. OBSERVATIONS
X CALCULATED POINTS
50
100
400
450
500
Fig. 8.
150 200 250 300 350
DIAMETER OF FOLLICLE, n.
Same as Fig. 7. Data on the rat. (From the Proceedings of the Royal
Society.)
preparations are not comparable phenomena. Furthermore,
dwarf mice pituitaries can stimulate ovarian growth in im-
mature recipients (Smith and MacDowell, 1931) yet their
THE GROWTH OF THE OVUM 35
follicles do develop to the stage of antrum formation. The
absence of eosinophile cells in the pituitaries of dwarf mice
may, however, indicate the absence of a necessary link in the
chain of steps involved in the hypophysis-gonad relationship.
Whatever the effect of ovarian maturation upon the pi-
tuitary may be, it is plain that no follicular response to pitui-
tary hormones occurs until the time when full sized ova are
present. Does this mean that the maturation of the follicle
120
iioH
100
^.90
^ 80
^ 70
O 60
H 50
H
S
^ 30
(a) . /
(b)
. OBSERVATIONS
X CALCULATED POINTS
100 200 300 400 500 600 700 800 900 1000
DIAMETER OF FOLLICLE, (jl.
Fig. 9. Same as Fig. 8. Data on the ferret. (From the Proceedings of the
Royal Society.)
is dependent initially upon some influence of the ovum, or
is the simultaneous development of the ovum to full size
and follicular growth to stimulable size a coincidence only?
The ovum may grow to full size without an investiture of
follicle cells as attested by the frequent presence of such
ova in the ovaries of dwarf mice (Pincus, unpublished data).
On the other hand, anovular follicles do occur in mammalian
ovaries (League and Hartman, 1925) though those of large
size represent follicles with completely resorbed ova (Engle,
19276). It is interesting to note also that frequent produc-
tion of anovular follicles from the germinal epithelium takes
place in senile rats (Hargitt, 1930).
36
THE EGGS OF MAMMALS
That the growth of the folHcle beyond the antrum stage
is independent of the growth of the ovum is amply evident
from the data presented by Brambell (1928), Parkes (1931)
90
P 70
fe 60
O
g 50
H
W
S 40
<
SO
20
(b)
• OBSERVATIONS
X CALCULATED POINTS
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
DIAMETER OF FOLLICLE, fx.
Fig, 10. Same as Fig, 7, Data on the pig, (From the Proceedings of the Royal
Society.)
and Pincus and Enzmann (19366). In Table III are presented
the data collected by Parkes on the relation of ovum size
to body weight and follicle size in seven species of mammals.
150
-
/
D
— o
^
6
7
8
9
100
~
/
/
A
50
t
^
A
n
h
1
1
1
I
1
1
1
1
200 400
600
800
1000 1200 1400 1600 1800
DIAMETER OF FOLLICLE
Fig, 11. Same as Fig. 7. Data on the rabbit. The lower curve represents
ovum diameter plotted against folhcle diameter for the nine types of follicles
(see Plate III) distinguished by Pincus and Enzmann, 19366.
Figures 7 to 10 relate the various diameters of ova to the
diameters of the enclosing follicles. In the rabbit, Pincus
and Enzmann (19366) have identified 9 types of follicles
each distinguished by characteristic features of the develop-
ing ovum, granulosa and theca (see Plate III). When the
k^ '9
Fig. 1
Fig. 2
Fig. 3
Fig. 7
Fig. 8
Fig. 9
Plate III. The development of the folhcle and ovum in mature rabbit does.
Fig. 1, Type 1 folhcle to the left, type 2 follicle to the right. Nuclei in late con-
densation of prophase. Fig. 2, Follicle type 3. One row of follicle cells. Fig. 3, Fol-
hcle type 4. Two rows of follicle cells. Fig. 4, Follicle type 5. Many rows of follicle
cells. Nucleus migrating to periphery. Fig. 5, Follicle type 6. Antra forming.
Fig. 6, Follicle type 7. Numerous antra. Fig. 7, Follicle type 8. Ovum suspended
in "spider web" of follicle cells. Corona formed. Fig. 8, Follicle type 9. Last
preovulatory stage. Fig. 9, Showing position of various follicle types beneath the
germinal epithelium.
37
38
THE EGGS OF MAMMALS
mean ovum diameters are plotted against the mean follicle
diameters (see Figure 11) the resulting curves essentially
resemble those illustrated in previous figures, the full ovum
size being attained in follicles of type 5 which just precede
antrum formation.
TABLE III
Size of the Graafian Follicle at Various Stages of Its Life-History
(From Parkes, 1931)
Species
1
Approximate
Weight of
Young Adult
Female
2
Diameter
OF Ovum
3
Diameter of
Follicle
WHEN Ovum Is
Fully Grown
4
Diameter of
Follicle
when Antrum
Appears
5
Diameter of
Follicle at
Ovulation
gm.
M
/^
M
mm.
Mouse
2 X 10
70
125
200
0.55
Rat
1.2 X 102
63
160
200
0.90
Ferret
5 X 102
108
170
230
1.4
Rabbit
2 X 103
84
145
250
1.8
Baboon
1.2 X 10^
83
180
310
6.0
Pig
5 X 10^
76
300
400
8.0
Cow
4 X 10^
—
—
—
15.0
The data plotted in this manner give no indication of the
absolute rate of growth of ova though the relative growth rates
may be deduced from the rising segment of the curves drawn
to these data. These first segments are plotted in Figure 12,
wherein it might be deduced that the ferret ovum grows at the
most rapid rate, the pig ovum at the slowest rate, if com-
parable rates of follicular growth occur in the various species.
If it be assumed that the various types of follicles de-
scribed by Pincus and Enzmann represent developments
occurring at equal time intervals then the lower curve of
Figure 11 may be taken as a representation of the growth
curve of the ovum. The sigmoid shape of this curve is in
fact reminiscent of general growth curves. It cannot be
taken as a true growth curve, however, until the time
necessary for the development of each type of follicle is
accurately known. Such information might very well be
obtained from ovaries subjected to x-irradiation and ex-
amined at various intervals after exposure.
THE GROWTH OF THE OVUM
39
110-
100
90-
60-
50
The data of Aral (1920a) give a slight indication of the rate
of growth of ova since his tables show no ova above 20 /z
in diameter in 1 day rats, the first appearance of 20 to 40 ijl
in ova in 3 day rats, the first appearance of 40 to 60 /jl ova
in 10 day rats, and ova over 60 ijl in rats over 15 days of age.
Thus it may be inferred that growth to full size is attained
in a little over two weeks. Whether this time is taken also
in adult animals is not
known exactly, but
the minimum period is
at least ten days since
irradiated mice pro-
duce fertile eggs up to
ten days after irradia-
tion (Parkes, 1926-
27). This unplies that
there is a sensitive
period to x-irradiation
in young ova. Mar-
shak (1935) has shown
that the pachytene
stage of meiosis is es-
pecially sensitive to
x-rays, and young ova
enter into a modified
pachytene shortly
after leaving the ger- gj^^h^of
minal epithelium. mammals.
Not all ova grow to '^^^"^^^•^
mature size. This is evident at once from Aral's data which
show that an average of 0.8 per cent of the ova under 20 /x
in diameter attain a diameter greater than 60 fi, and only
2.7 per cent reach 20 to 40 jj. in diameter. The factors con-
cerned in the atresia of young ova are as unknown as those
determining their growth.
The absolute size attained by mature ova varies from
species to species, but the limits are rather narrow (espe-
o
o
w
H
W
O 40-
30
20
1 1 1
100 200 300
DIAMETER OF FOLLICLE, n-
Same as Fig. 7, showing comparative
the ovum in the various species of
(From the Proceedings of the Royal
40
THE EGGS OF MAMMALS
TABLE IV
Estimates of the Diameter of Full-Grown Mammalian Ova
(From Hartman, 1929)
Animal
Monotremata
Platypus
Echidna
Marsupialia
Dasyurus
Didelphis
Edentata
Armadillo
Cetacea
Whales
Insectivora
Mole (Talpa)
Hedgehog (Erinaceus)
Rodentia
Mouse
Rat
Guinea pig
Lagomorpha
Rabbit
Carnivora
Dog
Cat
Ferret
Ungulata
Horse
Sheep
Goat
Pig
Cheiroptera
Bat
Lemurs
Tarsius
Primates
Gibbon
M. rhesus
Gorilla
Man
^losT Pkobable Size
OF Egg in Micra
2.5 mm.
3.0 mm.
240
140-160
80
140
125
100
70- 75
70- 75
75- 85
120-130
135-145
120-130
120
135
120
140
120-140
95-105
90
110-120
110-120
130-140
130-140
cially in the placental mammals) when comparison is made
with other vertebrates or inveterbrates. Hartman (1929)
has extensively reviewed the available data on fixed and
living material and has estimated the average size of the
Ii\dng OA^m for a number of species making allowance for
the degree of shrinkage in fixed preparations. His estimates
THE GROWTH OF THE OVUM 41
are given in Table IV. Subsequent measurements on living
ova have proved these estimates to be on the whole remark-
ably exact. An excellent brief account of the comparative
morphology of living mammalian ova in several species is
given by Streeter (1931).
CHAPTER IV
THE DEVELOPMENT AND ATRESIA OF
FULI^GROWN OVA AND THE PROBLEM OF
OVARIAN PARTHENOGENESIS
Even when the ova have attained maximum size a major-
ity of them are destined to degenerate. We have already
mentioned that Allen, Kountz and Francis (1925) estimated
that only 14 per cent of the
70
60
50
30
20
10
/
\ ATRETIC
'
\ FOLLI
\
CLES
\ PSEUDO
Y
S MATURATION
/
^^ SPINDLES
/
\
/
\
f
\
\
\
N
— ""rp.s.
medium sized follicles of the
pig ovary attain maturity.
Engle (19276) finds that in
the mouse the percentage of
atresia among follicles with
antra varies with the stage
of the oestrus cycle, the
maximum percentage of 86
per cent being recorded at
the cornified cell stage.
While the percentage of
atretic follicles with mature
ova was highest at the
oestrus stage the maximum
number was observed at the
beginning of the dioestrus.
This is obvious from the
data of Table V and Fig-
ure 13 which summarize the
data on 50 ovaries from non-
pregnant mice taken at four stages of the cycle. These data
include small atretic follicles as well as antrum-containing
folUcles, but the fact that the data for pseudomaturation
spindles (which occur only in full sized ova) parallel those
42
CORN L.E.l L E.^
STAGE OF CYCLE
Fig. 13. Showing the number of atretic
folUcles and pseudomaturation spindles
in the median ovary at four stages of the
oestrus cycle in the mouse. (From the
American Journal of Anatomy.)
OVARIAN ATRESIA AND PARTHENOGENESIS 43
for follicles indicates that the total number of atretic mature
ova reach their maximum in early dioestrus shortly after
ovulation. This is doubtless due to the continued formation
TABLE V
The Degree of Atresia of Ovarian Follicles of the Mouse at Four
Stages of the Oestrus Cycle. (From Engle, 19276)
Stage
OF
Cycle
Median
OF
Spindles
Average
OF
Spindles
Range
OF
Atresia
Median
OF Total
Atresia
Average
OF Total
Atresia
Range of
Total
Atresia
Corn
16
18.5
6-40
59
60
36- 85
LEI
26
28.4
7-51
71
77.9
50-129
LE2
11
11.2
5-17
36
42.1
29- 63
13
12
0-26
39
39.8
15- 70
of antrum-containing foUicles at a fairly high rate for a
short time after ovulation.
We have seen that ovogenesis continues during preg-
nancy. Engle's data dem-
onstrate that the formation ^.
30
and atresia of full-grown ^^
ova also occurs during preg
nancy, for he observed an t.§
appreciable number of c^gio
pseudomaturation spindles
in ovaries taken during the
first 43/2 days of pregnancy.
These data are summarized
in Table VI and Figure 14.
It is notable that both the
total amount of atresia and
the atresia of mature ova is
ATRETIC FOLLICLES
PSEUDO-MATURATION
>-... SPINDLES I ^--''
•---.i \-^
STAGE OF DEVELOPMENT
-Fig. 14. Showing the number of
, , , i ii • • 1 atretic folhcles and pseudomaturation
less throughout this period spindles in the median ovary at four
than during the period of stages in early pregnancy in the mouse.
1 i J i J- • (From the American Journal of Anat-
least destruction m non- ^^^^y^
pregnant mice. Unfortu-
nately, Engle does not give the percentages of atresia during
early pregnancy.
The presence of cycles of atresia and growth in animals
44
THE EGGS OF MAMMALS
other than the mouse has already been noted (Evans and
Swezy, 1931). According to Asami (1920) the rabbit ex-
hibits a constant rate of folUcular atresia before and after
TABLE VI
The Degree of Atresia of Ovarian Follicles of the Mouse
Stages of Early Pregnancy. (From Engle, 19276)
AT Four
Stage of Tubal Ova
Median
OF
Spindles
Average
OF
Spindles
Range
OF
Spindles
Median
OF Total
Number
Atresia
Average
OF Total
Number
Atresia
Range of
Total
Number
Atresia
To 2 pronuclei
2 to 4 blastomeres
Morula
Blastocyst
5
2
3
6
5
2.5
5.6
7.1
0-15
0- 7
2-20
3-14
22
17
13
15
23
18
16.5
17.3
11-41
10-31
7-38
14-28
pregnancy. Pincus and Enzmann (19366) found that the
younger folUcles (types 1, 2 and 3 — Plate III) of the rabbit
show a much lower percentage of atresia than the larger
follicles.
The atresia of mature ova can be prevented by pituitary
hormones. This is deduced from the phenomenon of super-
ovulation observed in animals receiving pituitary implants
(Smith and Engle, 1927; Smith, 1932). These authors
describe, for example, the presence of 49 ova in the tubes of
a mature mouse receiving anterior lobe implantations. An
adult mouse produces from six to twelve corpora lutea at
an ovulation, the absolute number varying with weight of
the mouse, the number of previous pregnancies, and certain
genetic factors (MacDowell and Lord, 1925; MacDowell,
Allen and MacDowell, 1929). In Smith and Engle's mice the
largest number of ova ever found in one tube of a normal
mouse was seven, and in an immature mouse showing super-
ovulation a maximum of 48 ova was observed in a single
tube. Thus the maximum number normally found in one
tube is 14.5 per cent of the maximum number super ovulated.
Furthermore, if we assume from MacDowell's data that 9
is roughly the number of ova normally ovulated this is
18 per cent of the 49 superovulated in the adult mouse.
These percentages agree with the estimations of per cent
OVARIAN ATRESIA AND PARTHENOGENESIS 45
of antrum-containing follicles maturing. The paucity of
antrum-containing follicles and reduction of atresia is di-
rectly noted by Smith and Engle. Finally, the ovulated
ova are fertilizable although Engle (19316) found evidences
of the degeneration of a number of them in the fallopian
tubes.
An interpretation of the foregoing data is that normally
only a limited amount of pituitary secretion is available to
the ovary and consequently only a certain percentage of the
ova are able to obtain the amount necessary to prevent
their atresia, whereas in animals receiving large amounts of
pituitary hormones from implants an abnormal number of
ova have available sufficient amounts of atresia-suppressing
hormones. It cannot be decided, however, whether the
effect on the ova is directly exerted by these hormones, or
whether the stimulated follicle tissue produces substances
ensuring normal ova, or whether some extraovarian sub-
stance released into the circulation by pituitary stimulation
reacts upon the ova.
Loeb (1917; see also Meyer, 1913) has indeed suggested
that the ovum itself is the controlling factor in follicle
development citing the frequent presence of mitoses in fol-
licle cells adjacent to the ovum as well as certain histological
evidence that the cumulus oophorus develops under the
influence of the ovum (Walsh, 1917). Allen and his collab-
orators (1924) also maintained that the ovum is the dynamic
center of the follicle apparently on the assumption that the
mitosis-inducing action of oestrin upon vaginal and uterine
epithelium is reflected in the higher mitosis rate in cells
adjacent to the ovum becausB the ovum either produces
oestrin or induces oestrin formation. In the opossum the
presence of many atretic ova is correlated with prolongation
of the dioestrus interval (Hartman). This supposed oestrin-
ogenic action of the ovum has, however, been largely con-
troverted (1) by the discovery of oestrin in corpora lutea
as long as two weeks after ovulation (see Allen, 1932) and
(2) by the observation that oestrin is produced in x-rayed
46
THE EGGS OF MAMMALS
ovaries lacking ova (Parkes, 1926-27). This evidence, how-
ever, does not prove that normally oestrin-production may
not be under the control of the action of pituitary hormones
upon the ovum itself.
In fact, aside from the presumable atresia-inhibiting in-
fluence, there seems to be only one other clearly evident
influence of pituitary hormones upon the activities of the
»^sfr'*-T^""' ?^
Fig. 15. Ovum removed from a preovulatory follicle of
an unmated rabbit showing the vesicular nucleus. (From
the Journal of Experimental Medicine.)
ovum. That is that the production of the first polar body is
dependent upon stimulation by pituitary hormones.
Since this phenomenon is of some consequence to any
discussion of the activation of mammalian eggs the writer,
in collaboration with Dr. E. V. Enzmann (Pincus and Enz-
mann, 1935), has undertaken an examination of the mecha-
nism of polar body formation in the rabbit ovary. The rabbit
was chosen for these experiments because it ovulates only
after copulation and the ova are liberated regularly between
93^ and 103^ hours after copulation (see Heape, 1905;
Walton and Hammond, 1932; Pincus, 1930; Pincus and
Enzmann, 1932). Furthermore, the mature ova form polar
bodies only after copulation. According to Heape (1905)
OVARIAN ATRESIA AND PARTHENOGENESIS 47
two polar bodies are formed in the ovary by 9 hours after
copulation. Our observations indicate that only the first
polar body is given off in the ovary and then the metaphase
plate of the second polar spindle is formed. Robinson (1918)
observed in the ferret, which also ovulates only after copu-
lation, that only the first polar body is given off in the ovary
some time after copulation.
^ - i
m
^M
Fig. 16. Ovum removed from a ripe follicle of a rabbit doe at two hours
after copulation. Note beginning of chromatin condensation. (From the
Journal of Experimental Medicine.)
Before copulation occurs the mature ovum contains a
single large vesicular nucleus about 30 microns in diameter
(Figure 15; see also Plate III, Figs. 4 and 5). At two hours
after copulation signs of change are partially evident: some
of the ripe ova show the beginnings of tetrad formation in
the nucleus but the nuclear membrane is still intact (Fig-
ure 16). By four hours after copulation the tetrads of the
first polar spindle are formed and the nuclear membrane
is ordinarily dissolved (Figure 17). The metaphase plate
has a diameter of a little over 10 microns. The first polar
48 THE EGGS OF MAMMALS
body is given off and the second polar spindle formed at or
shortly after 8 hours post coitum (Figure 18). The follicle
enlarges during this period also, the first signs of follicular
development being evident at two hours after copulation.
An exactly similar sequence of events occurs when prolan
(pregnancy urine extract) or anterior pituitary extracts are
injected.
-#
^mf9 ^^^
Fig. 17. Ovum from follicle of ral)l)it don taken 4 hours after copulation.
Formation of metaphase plate and dissolution of nuclear membrane. (From
the Journal of Experimental Medicine.)
It has been definitely established that prolan and anterior
pituitary hormones cause ovulation when injected into the
rabbit (Bellerby, 1929; Friedman, 1929). The ovulation
occurring after copulation occurs because of the increased
level of pituitary hormones secreted into the blood. This
level is increased by nervous stimulation of the pituitary
consequent on the orgasm. It has been shown by Deansley,
Fee and Parkes (1930) that hypophysectomy within one
hour of copulation prevents ovulation in the rabbit (see also
Smith and White, 1931), and McPhail fl933) has demon-
OVARIAN ATRESIA AND PARTHENOGENESIS 49
strated similarly that the critical period of secretion increase
in the ferret occurs during the first hour of coitus. It seems
evident, therefore, that pituitary secretions are responsible
for the activation of the egg resulting in the formation of
the first polar body and the second polar spindle. Further-
more, certain observations of Hinsey and Markee (1933)
indicate that the threshold for activation is lower than the
^
'" ™
i
Fig. 18. Ovarian ovum of rabbit doe mated 9 hours previously. First polar
body and second polar spindle. (From the Journal of Experimental Medi-
cine.)
threshold for ovulation. They observed that ovulation does
not occur in large sized (2.6 kilograms and over) hypophy-
sectomized rabbit does if prolan injection is made more than
four hours after hypophysectomy. And in small sized hy-
pophysectomized does (less than 2.3 kilograms) prolan ovula-
tion never occurs. Nonetheless in all non-ovulating does
polar body formation took place. Friedgood and Pincus
(1935) found that stimulation of the cervical sympathetic
of the rabbit resulted in maturation phenomena in those
preovulatory follicles which failed to liberate ova. The
sympathetic nerves presumably stimulated in these cases
50 THE EGGS OF MAMMALS
the secretion of sub-ovulatory amounts of hormone from
the anterior pituitary. Finally, Pincus and Enzmann (1935)
found definite ovum maturation with as little as 34 the
minimal ovulating dose of maturity hormone.
In the ovaries of rabbit does which have copulated and
then received pituitary injections within six hours after
copulation the writer has observed the accelerated ripening
of a new set of follicles and the formation of the first polar
body. In these rabbits no accessory ovulation occurred
though the pituitary extract dosages were at least two to
three times greater than those necessary to cause ovulation
in unmated does. The absence of ovulation indicates pre-
sumably that the expulsion of ova can occur only from full
sized follicles, whereas the activation processes may be in-
itiated in ova whenever a sufficiency of pituitary hormones
are available. It should be neted, however, that the nuclear
activity occurred only in medium sized follicles and never
in follicles without antra or with small antra forming. Since
the ovum in the rabbit grows to some extent after antrum
formation (see Figure 11) it is possible that functional matu-
rity is attained at some time after antrum formation. A new
crop of follicles begins to mature in the mated rabbit, and
may certainly be stimulated to ovulate by the 4th day of
pregnancy as Wislocki and Snyder (1931) have demonstrated
by producing superfetation at that time with simultaneous
pituitary extract and sperm administration. It is evident,
therefore, that any attempt to dissociate in vivo the processes
involved in polar body formation and those involved in
ovulation depends in the mature rabbit upon hormone ad-
ministration during the very short interval of time following
copulation in the hope that active substances reaching the
medium sized follicles will differentially affect foUicular
growth and ovum maturation.
Since the pituitary secretes a thyroid-stimulating as well
as a gonadotropic hormone it is possible that maturation
(and ovulation) is due directly to thyroid activity and only
indirectly to pituitary stimulation. Pincus and Enzmann
OVARIAN ATRESIA AND PARTHENOGENESIS 51
(1935) tested this possibility by injecting crystalline thyroxin
and thyroprotein into rabbit does on heat. In no instance
did ovulation occur but large doses of thyroxin did initiate
follicular atresia and a limited degree of o\aim maturation.
Again we see that atresia-inducing conditions also initiate
maturation. The common feature of atretic follicles and
preo\ailatory follicles is an isolation of the ovum from its
connections with the follicular epithelium.
It is safe to conclude from the foregoing analysis that the
formation of the first polar body in the rabbit ovary (and in
the ferret's also) is dependent upon an increase of pituitary
hormones in the circulating blood. It happens that in all
spontaneously ovulating mammals except the dog the forma-
tion of the first polar body occurs in the ovary. Even in the
dog (Evans and Cole, 1931) certain signs of nuclear matura-
tion are observable in ovarian eggs. It is natural to infer
that in spontaneously ovulating animals the pituitary level
reached during oestrus is normally sufficient to induce ovula-
tion as well as polar body formation.
Now it is notable that the atresia of ovarian eggs is often
initiated by the formation of a maturation spindle. We
have noted that Engle has designated the spindles of ova
destined to atrophy as '^pseudomaturation" although there
is no evidence that they are in fact typically unlike those
observed in normally maturing ova. Such spindles are ob-
served only in ova of full size. Measurements of spindle
containing ova in mouse ovaries give an average maximum
diameter of 70 microns, and mature ova with vesicular
nuclei had an average of 69 microns. The writer has also
made careful examination of a large number of rabbit ovaries
and has never observed typical spindles in immature eggs.
What ordinarily occurs is a complex fragmentation of the
chromatin (see Figure 19). That the spindles are the indices
of impending atresia is indicated by the observation that
when they are at a maximum the total follicular atresia is
also at a maximum (see Figures 13 and 14). A possible in-
terpretation of their presence may be that they occur as a
52
THE EGGS OF MAMMALS
result of pituitary hormone action and the subsequent atresia
of the ova containing them occurs because these ova are not
Uberated and fertihzed. " Pseudomaturation " spindles have
not been reported in hypophysectomized animals although
atresia has.
It has long been the contention of certain observers of
ovarian atresia that the apparent parthenogenetic develop-
t^ 'Mm:
'»!.
Fig. 19. Atretic ovum from type 3 follicle in the rabbit.
Note fragmentation of cytoplasm and chromatin.
ment of ova destined never to be liberated is simply an
incident of the process of degeneration and is not in fact
true parthenogenesis (Hensen, 1869; Balfour, 1882; Sobotta,
1899; Janosik, 1897; Bonnet, 1899; Rubaschkin, 1906; Ath-
ias, 1909; Kingery, 1914; Kirkham, 1916; Stockard and
Papanicolou, 1917; Addison, 1917; Long and Evans, 1922;
Clark, 1923; Engle, 19276; Kampmeier, 1929). These in-
vestigators have observed varied types of fragmentation of
OVARIAN ATRESIA AND PARTHENOGENESIS 53
egg nucleus and cytoplasm, most of which cannot be con-
sidered the result of true cleavage processes though in some
instances a remarkable resemblance to cleaved ova is at-
tained (see Plates IV and V). Another group of investigators
generally admit that complex pseudoparthenogenetic frag-
mentation occurs, but claim that a varying number of ova
enter into true parthenogenetic development (Pfluger, 1863;
Flemming, 1885; Paladino, 1887; Lowenthal, 1888; Schott-
lander, 1891; Henneguy, 1893; Grusdew, 1896; Rabl, 1898;
Gurwitsch, 1900; Spuler, 1900; Van der Stricht, 1901; Loeb,
1901, 1905, 1911, a and b, 1912, 1915, 1923, 1932; Newman,
1912, 1913; Sansom, 1920; Haggstrom, 1922; Courrier and
Oberling, 1923; Courrier, 1923; Branca, 1925; Bosaeus,
1926; Lelievre, Peyron and Corsy, 1927). The resolution of
such alternative points of view depends first of all upon a
clear definition of what parthenogenesis is and secondly upon
the interpretation of the ovarian structures designated as
embryonic.
If by parthenogenesis is meant the development of a
mature individual from an unfertilized egg then it is at once
certain that parthenogenesis does not take place in mamma-
lian ovaries. If, on the other hand, a cleavage of the ovum
with an equational division of the chromosomes is the cri-
terion then there is some evidence (Sansom, 1920; Branca,
1925; Engle, 19276) that occasionally parthenogenesis occurs
in ovarian eggs (see Plates IV and V). Certainly it is not
permissible to consider as parthenogenesis an exact repro-
duction of events taking place in the fertilized egg, since it
is well known, for example, that parthenogenetic individuals
arise from ova in which second polar body formation is
suppressed.
It seems appropriate, in seeking an understanding of the
physiological processes occurring in developing eggs, to dis-
tinguish between parthenogenesis and activation. A definite
series of physical and chemical events ensue in eggs treated
by agents inducing parthenogenesis. An apparently iden-
tical set of changes occurs at fertilization. This process
54 THE EGGS OF MAMMALS
which Needham (1932) has designated ''an opening of doors''
in the cell initiates the development of the ovum and makes
of a static cell one capable of transformation. What happens
subsequent to the activation process is often independent
of the process itself. The probability of cleavage and the
formation of a complete individual depends in part on the
nutritional environment and the chromosome constitution
of the activated egg.
The activation process in non-mammalian ova has been
described in physico-chemical terms (see J. Loeb, 1913;
F. Lillie, 1919; Just, 1928; Runnstrom, 1933; Whit aker, 1933;
R. Lillie, 1934). There exists no similar information partic-
ularly for the ovarian eggs of mammals. The only estab-
lished index of an activation of ovarian eggs is the described
formation of the first polar body. It is conceivable that this
represents the first step in an activation process that would
go to completion if conditions were propitious. Perhaps the
same pituitary stimulus that induces polar body formation
might cause the formation of a cleavage spindle. The first
cleavage spindles observed by Branca (1925) may then be
considered the result of an activation process carried to
completion because adequate pituitary stimulation was avail-
able. On this basis the liberation of ova from the ovary
results in such a change of environment that the stimulus to
completion of activation is ordinarily no longer available.
Similarly mature ova retained in the ovary at the time of
ovulation ordinarily degenerate either because the proper
type of pituitary hormone is not active (c/. Hisaw's concep-
tion of the alternative action of follicle stimulating and
luteinizing hormones) or because of the partition of the
active hormone to other tissues {e.g., corpora lutea).
We may consider two further alternative explanations
of the activation of ovarian eggs. It is possible that activa-
tion occurs in the ova of degenerating follicles because
(1) the breakdown of cells near the ovum results in the re-
lease of activating substances or (2) the initial stages of
atresia in the egg cytoplasm frequently involve structural
OVARIAN ATRESIA AND PARTHENOGENESIS 55
changes in the egg cytoplasm which are identical with those
changes occurring during normal activation.
According to the first of these two alternatives cell divi-
sion stimulating substances are released as break-down prod-
ucts (Gutherz, 1925). That such substances are actually
formed by mammalian cells has been attested by the study
of the growth of tissue cultures (Carrel, 1924; Fischer, 1925)
where they have been given the name trephones. Further-
more, signs of atresia in theca and granulosa cells are cyto-
logically evident before signs of ovum breakdown. It has
never been conclusively demonstrated, however, that treph-
ones can activate ova (but see Haberlandt, 1922). On
the other hand, it is conceivable that regardless of trephone
action, the degeneration of follicle cells leads to a stimulating
concentration of cytolizing substances {e.g., fatty acids which
are known to act as activating agents) or even to a sufficient
hypertonicity in the region of the ovum.
The second of these alternatives implies that ^Hhe open-
ing of doors" occurring in normal activation is an aspect
of degeneration. Atresia certainly involves changes in the
colloidal structure of cells, and we have pointed out {vide
supra) that definite changes in cortical structure mark the
activation process. It is interesting, therefore, to note that
the cytological appearance of the cytoplasm of retained ova
with spindles is markedly similar to that of fertilized eggs.
Thus the cytoplasm of unfertilized eggs have upon fixation
a rough coarsely reticular appearance (see Figure 15 and
Plate III, Fig. 4), whereas retained ova with spindles, like
normally activated or fertilized eggs, have a uniformly
granular cytoplasm (Figure 18).
Whether stimuli from degenerating follicle cells or endog-
enous structure changes are involved, it is evident that
these factors are in turn conditioned by the supply of avail-
able hormone. Insufficient pituitary hormone results in the
creation of ovum activating conditions. This is on the face
of it, in direct contradiction of the first hypothesis which
states that a supraliminal supply of hormone may also
56
THE EGGS OF MAMMALS
initiate activation. But this contradiction may be resolved
if we consider that the same conditions may be created by
either active pituitary stimulation or absence of it.
It has been shown that pituitary hormones themselves
TABLE VII
The Development of Ovarian Eggs of the Rabbit in Media Containing
Various Hormone Preparations. (From Pincus and Enzmann, 1935)
Number
Time of
OF
Medium
Results
CULTURING
Cultures
20 min.
14
Ringer-Locke +
1 drop beef
pituitary
Vesicular tetrads formed in
all cases
2 hrs.
11
Ringer-Locke -|-
2 drops beef
pituitary
In some cases vesicular tet-
rads and some free tetrads
were formed. Some formed
polar bodies
24 hrs.
9
Ringer-Locke -f-
1 drop maturity
hormone
Vesicular tetrads in all cases
except 3 which had free
tetrads
25 hrs.
4
Ringer-Locke -f
2 drops maturity
Vesicular tetrads in all cases
A.
hormone
25 hrs.
7
Ringer-Locke +
3 drops maturity
hormone
Vesicular tetrads, free tet-
rads, structures resembling
fusion nuclei
2 hrs.
18
Ringer-Locke
Vesicular tetrads and free
tetrads
4 hrs.
3
Ringer-Locke
Free tetrads
6 hrs.
3
Ringer-Locke
Rudiment of first polar spin-
dle
20 hrs.
IG
Ringer-Locke
Vesicular tetrads, free tet-
rads, fusion nuclei
24 hrs.
6
Plasma + 1 drop
thyroxin
22 hrs.
4
Plasma + 3 drops
thyroxin
22 hrs.
3
Phisma -|- 4 drops
All cultures showed about the
B
thyroxin
same phenomena which in-
24 lirs.
8
Plasma -|- 6 drops
thyroxin
cluded tetrad formation in
all cultured eggs. In some
24 hrs.
4
Plasma -|- 8 drops
thyroxin
of the cultures polar bodies
formed, or the vesicular
20-24 hrs.
22
Plasma -|- 2 drops
Ringer-Locke sol.
membrane dissolved
. 20-24 hrs.
8
Plasma -|- 6 drops
Ringer-Locke sol.
OVARIAN ATRESIA AND PARTHENOGENESIS 57
do not act directly upon the ova (Pincus and Enzmann,
1935) by experiments in which ovarian ova with vesicular
nuclei were cultured in media containing various pituitary
extracts. The data of these experiments are summarized
in Table VII-A. They show that in both the extract-
containing media and the extract-free media maturation
proceeds at about the same rate. Furthermore thyroxin
which causes a certain degree of maturation when injected
in vivo (see page 51 above), causes in vitro no further degree
of development than thyroxin-free controls (Table VII-B).
The isolation of the ova from the normal follicular environ-
ment is sufficient to initiate activation. This implies that
in preovulatory follicles maturation is caused by either
(1) the mechanical separation of the ovum and its corona
or (2) the removal of an inhibiting influence. Mechanical
separation undoubtedly occurs (c/. Plate III, Figs. 8 and
9), but one cannot estimate the exact degree of isolation
necessary to initiate maturation, for it is certain (Pincus and
Enzmann, 1935) that maturation is initiated in ova still
having strands connecting them to the follicular epithelium.
In certain forms {e.g., man) the ova remain embedded in
the cumulus mass till just before ovulation and the corona
forms late. It is notable that Allen, Pratt, Newell and
Bland (19306) were able to obtain only one maturation stage
in some two hundred ova recovered from 3 to 20 mm. fol-
licles. The writer (unpublished data) has observed one
maturation occurring in a primate ovarian ovum, but when
primate ovarian ova are cultured in vitro considerable nu-
clear activity occurs. During the first stages of pituitary-
induced maturation in the rabbit a secretion of secondary
liquor folliculi is observed (Pincus and Enzmann, 1935).
This secretion may remove an activation-inhibiting influ-
ence. The maturation observed in ova of atretic follicles
may be due to a similar sort of secretion rather than to
simple isolation of the ovum from its follicular epithelium.
On the basis of the foregoing considerations one might
conceivably encounter occasional evidences of activation
Fig. 1
Fig. 2
^ \ ^
r:?
Fig. 3
Fig. 4
Fig. 5
'X^«
Fig. 6
Plate IV. Various stages in the development of the mature oocyte.
(From the Archives de Biologie.)
Fig. 1, First maturation spindle — guinea pig. Fig. 2, Binucleated ovum, chro-
mosomes oriented for the metaphase of a mitosis — guinea pig. Fig. 3, Binucleated
ovum with formed maturation spindles — mouse. Fig. 4, Multinucleate cytoplasm —
mouse. Figs. 5 and 6, Typical uninucleate cleaved ovocytes. Fig. 6 shows deuto-
plasmic extrusions — guinea pig.
58
OVARIAN ATRESIA AND PARTHENOGENESIS 59
where alterations in normal hormone balance occur which
are sufficient to cause a preponderating activation stimulus.
Such may in fact be the basic cause of certain undoubtedly
normal early development in ovarian eggs reported by a
number of observers. In Plate IV, Figures 1 to 3 and Fig-
ure 1, Plate V, are presented various stages of pre-cleavage
development found in ovarian eggs. The multinucleate con-
dition of the egg of Figure 4 may be due to chromatin
fragmentation, but the cleavages of the eggs of Figures 5
and 6 of Plate IV and Figures 2 and 3 of plate V are com-
pletely normal. It seems clear that at any one of these stages
definite atresia of the ovum may set in, preventing further
development. Similar arrests of development may occur in
parthenogenetically activated invertebrate ova if the acti-
vating treatment is not carefully controlled (c/. Loeb, 1913;
Just, 1928). Entrance into the cleavage process is likewise
dependent upon a rather nice balance of developmental
events. Furthermore, the processes involved in cleavage
may indeed be independent of the activation process.
Runnstrom (1933) has shown that sea-urchin eggs poisoned
by monoiodoacetic acid can be fertilized but that segmenta-
tion soon ceases and ordinarily just before the dissolution
of the nuclear membrane of the first cleavage division.
In later chapters we shall discuss further the problems
involved in parthenogenetic activation. Now it is sufficient
to indicate that there is a probability of activation of ovarian
eggs but that a complete activation is dependent upon a
balance of events which must presumably be rarely attained
in the ovary. Even if the activation reaction proper occurs
and segmentation ensues the probabilities that post-cleavage
stages will be entered are made extremely small not merely
because of the physical limitations imposed by the structure
of the ovary, but because, as we shall demonstrate later
(Chapter IX), the growth stage of the embryo is entered
into only as the result of a definite hormonal stimulus during
the luteal phase, and conversely is definitely inhibited by
oestrin. It is therefore surprising that the blastula and
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Plate V. (Figs. 1 to 3 from the Journal of Anatomy; Figs. 4 to 7 from
the Archives de Biologie.)
Fig. 1, Line drawing of a section through an atretic foUicle of the mouse. First
mitotic anaphase. Cytoplasmic division not completed — mouse. Fig. 2, Typical
4-celled ovarian ovum — water vole. Fig. 3, Typical 2-celled ovarian ovum with
intact zona — water vole. Fig. 4, Early blastocyst of ovarian ovum — mouse. Fig. 5,
Many-celled blastocyst in ovarian ovum — guinea pig. Fig. 6, Multinucleate blasto-
cyst-like ovarian ovum — mouse. Fig. 7, Blastocyst-like ovarian ovum — guinea pig.
60
OVARIAN ATRESIA AND PARTHENOGENESIS 61
neurula-like formations, described by Courrier (1923) (see
Figures 4 to 7, Plate V) and Courrier and Oberling (1923)
and the atypical ovarian embryos observed by Loeb (1932)
should be found. The solution to the controversy concern-
ing their exact nature must await evidence as to the pos-
sibility of their formation by experimental means.
It can be seen that the chance of atretic degeneration
continually besets the ovarian egg. The evidence indicates
this process can be avoided only if sufficient pituitary hor-
mone is available to the ovary. There exists also the possi-
bility that atresia is endogenous in the sense that the ovum
as a cell attains a certain maximum degree of development
and then inevitably goes down hill. Only the sudden inter-
vention of ovulation and fertilization prevents this process.
Such a conception is scarcely amenable to experimental
verification chiefly because of the intimate association of
the ovum and its follicle. Furthermore, signs of ovum de-
generation are preceded by degenerative phenomena in the
granulosa cells. If the granulosa and corona cells act as
nurse cells to the ovum it is obvious that their behavior
must largely condition the behavior of the ovum. Often
the ovum becomes detached from the granulosa and corona
radiata and floats practically free in the liquor folliculi.
We do not know to what extent diffusion of a sufficiency of
nutritive substances through the liquor folliculi is possible.
The problem of the viability and senescence of the ovum
still awaits experimental attack.
CHAPTER V
METHODS EMPLOYED IN THE EXPERIMENTAL
MANIPULATION OF MAMMALIAN OVA
The first investigators of living tubal eggs (Barry, Cruik-
shank, Bischoff, Spee, et al.) used rather laborious methods
of dissecting the tubes (see Squier, 1932, for an interesting
historical discussion). The modern technique of securing
eggs from the fallopian tubes of most mammals is a fairly
simple one. Nonetheless, certain surprising differences in
the behavior of the obtained ova arise when exactly the
same methods are applied to two different species. Among
the laboratory mammals the rabbit is by far superior, and
for one very simple reason, namely, rabbit ova seem to
withstand the process of handling better than other ova.
Mouse, rat and guinea pig ova, for example, begin to frag-
ment very soon after removal from the tubes (Lewis, 1931;
Gilchrist and Pincus, 1932; Squier, 1932; and Defrise, 1933)
and to date it has been possible to observe at most one or
two cleavages in culture, whereas rabbit ova will go through
the whole course of cleavage and blastulation in vitro.
The long, fairly straight tubes of most mammals can
easily be washed through by a Ringer-Locke or similar bal-
anced salt solution. The writer has found that a Ringer-
Locke solution to which has been added an equal amount
of homologous blood serum is most useful. It is necessary
only to free the tubes of their mesenteric connections, and
if the tubes only are to be employed to cut them away from
the uterus. It is ordinarily best to cut off the uterus at
about one-half inch from the ampulla so that if washing
backward toward the fimbria is desired a certain length of
uterine lumen will be available for the guidance of the
washing pipette. When ova are to be washed downward
62
METHODS FOR THE MANIPULATION OF OVA 63
from the fimbriated end of the tubes a rather broad bored
capillary pipette is used; washing upward from the uterine
end requires a very fine pipette. The ova are washed into
Syracuse watch glasses and are easily observed under low
magnification of a dissecting microscope.
In animals like the rat, mouse and guinea pig with coiled
tubes a different procedure is followed. Here the coiled
tubes are cut into several fairly straight portions and are
squeezed with a pair of fine iris forceps or stroked gently
with blunt needles. The contents of the tubal lumen are
extruded and the ova are found among the cellular
debris.
Ova from the uterus are obtained simply by flushing the
uterine lumen with the washing fluid.
Allen, Pratt, Newell and Bland (1930a) describe a method
for obtaining human tubal ova without removing the tubes
or uterus. ^'The ovaries were examined as soon as possible
after the abdominal cavity was opened. In some instances
the findings at operation necessitated removal of the most
recently ovulating ovary and its tube was not justified, the
tube was flushed in situ and the corpus luteum alone re-
moved from the ovary. This method consisted of clamping
the cervix with a special clamp and injecting isotonic saline
solution directly into the uterine cavity from above by
hypodermic syringe while first one and then the other uterine
tube was gently pinched by the assistant. The injected
solution in most cases flowed back freely through the tube
and was collected in a series of watch-glasses held beneath
the fimbriated end. Apparently the development of valve-
Hke folds of mucosa at the tubo-uterine junction as described
for several mammals by Lee (1928) is not appreciable in
woman. Usually from 10 to 30 c.c. was flushed through each
tube. The most recent corpus was carefully excised from
the ovary, and since it is a transitory structure, without
sacrificing any considerable amount of ovarian tissue.
"It is believed that this method of flushing the tubes
in situ is harmless to uterus and tubes and opens up new
64 THE EGGS OF IMAMMALS
possibilities, not only for the recovery of human ova, but
also for checking the patency of tubes at operation.
''The tubes which could be removed were washed by direct
injection through either the uterine or the fibriated ends
after first trimming the tube carefully along the attachment
of the mesosalpinx. The trimming seemed advisable, for
otherwise when the tube was distended with injected fluid
it would often kink badly.
''A search for human tubal ova is sometimes complicated
by the follicle cells of the cumulus still surrounding the
specimens which make difficult clear observation and certain
identification. Although while fresh such specimens are
fairly transparent, it is often difficult to observe or measure
them accurately. Since it is probable that ova may remain
in the tubes for three or more days, degenerative changes
may be expected in a certain number of unfertilized tubal
ova. Also small masses or balls of cells are often encountered
in the tubes. These may originate in the peritoneal ca\'it.y,
be pinched ofT from the fimbria of the tube, or (in cases
where injected fluid is forced back through the tubes from
the uterine cavity) derived from cast-off endometrium.
Sometimes such cell balls contain structures which before
sectioning can easily be mistaken for ova. For this reason
unless an ovum is free from follicle cells or the cells of cumulus
are partly dispersed, it w^ould seem necessary that it be
sectioned before certain identification is possible. Further
check should also be made by histologic study of the most
recent corpus luteum."
In obtaining both unfertilized and fertihzed ova for cul-
ture in litro the use of a warm washing solution is preferable.
This is often practically difficult and rabbit ova at least are
not materially affected by handling at room temperature
over a period of several hours.
The usual methods of tissue culture have been employed
in the cultivation of mammalian ova. These include the
hanging drop with the ovum held in a plasma clot on a
coverslip over a fluid-free cavity; a plasma clot occupying
Mirrifons for tiik ma\ipi:latio\ of o\'a go
the total area under a raised coverslip; the CJarrf^I flask;
and the watch-glass teehnirjue in which the sterile watch
glass containing the culture rnediun^i is contained in a nnoist
chamber, iilood plasma or serum ordinarily form the basis
of the most successful culture media. I'he longest perirjd of
rfigular development of normally fertilized rabbit ova has
bef*n obtained by IxnvLs and Gregory (1929j who photo-
graphed the development of rabbit ova from the initial
cleavage stages through late blastocyst stages. They placed
the ova in homologous plasma upon glass slides. Pincus
(unpublished data) has obtained similar development by this
technique and also with ova grown in Carrel flasks. IxwLs
and Ilartman (\iy.V4) observed the development of a Macacus
rhesus ov^um from the 2-cell to the 8-cell stage using the
Ixwis and Gregory technifjue. The ova of the rat, moase,
and guinea pig have failed to de\'elop beyond one or two
cleavages with the use of a \'ariety of culture media. I'hus
Defrise 0933; used the following media for culturing rat
ova: (\) Ringer's solution, bufferr.'d or not with sodium
bicarbone; (2) Tyrode's solution, (\) Isotonic with NaCl,
milimol: fa) 120, (h) VM), ((■) 151, (B) K+ - Ca^+ - Mg"+
equilibrium on the basis of the triangular diagram of Loewe,
milimol: Ta; 2.05 KCL, 1.90 CaCU, 2.20 MgCF,, ^b; 5.63 KCl,
3.60 CaCU, 0.52 MgCla: the above solutions were used at
pll 6.8, 7.2, 7.6; f3j Tyrode's solution rXaCl: milimol
136 - KCl: 5.6 - CaCU: 2.16 - MgCU: 0.52 - XaHC03:
8.6 - CJI 1206:5.5) with the addition of gelatine 0.5 per
cent; (4) Tyrode's solution (as above) with the addition of
blood serum: faj 1/1, fb) 3/1; (o) Tyrode's solution (a.s
above) with the addition of plasma (heparin): (a) 1/1,
(b) 3/1; (6) pure blood serum of (a) pregnant female,
(b) male, (c) newborn; (7) plasma (secured from the heart
and mixed with heparin): (a) pregnant female, (b) male;
(8) spinal fluid (secured by suboccipital puncture) ; (9) foetal
hystolymph (secured by Mart.ino\itch's technique); HO)
uterine fluid (II oestral period): (a) pure, (h) with the ad-
dition of blood serum.
66 THE EGGS OF MAMMALS
In a few cases, in some of the above media, and especially
inthis:NaClmilimol 130 - KCl 2.65 - CaCU 174 - MgCls
1.18 - NaHCOa 8.6 - at pH 7.2 - drops III = blood-
serum drops II, one or two mitoses were obtained. The addi-
tion of small quantities of embryonic extract, of rat foUic-
uline, of extract of the anterior lobe of the hypophysis to
the medium (either solid or liquid, natural or artificial)
has not noticeably modified the culture results.
Squier (1932) using a less extensive variety of media was
similarly unsuccessful with guinea pig ova (see also Lewis,
1931).
The limitations of the ordinary methods of tissue culture
are discussed further in Chapter IX in connection with the
investigation of the normal physiological environment of
developing ova.
Nicholas and Rudnick (1933) have cultivated rat embryos
upon the chorioallantois of the chick, but ovum development
has not been studied. The embryos survive and differenti-
ate over a considerable period of time in the foreign environ-
ment.
Cinematography of developing ova has been undertaken
in a number of recent investigations. Standard motion
picture cameras adapted for microphotography are employed.
For a study of the comparative behavior of ova in vivo
and in vitro the writer has transplanted cultured ova into the
fallopian tubes of rabbit does (see Pincus and Enzmann,
1934). The operative technique requires the use of a light
anaesthesia, e.g., either ether preceded by atropine sulphate
injection to inhibit excessive mucous secretion or simple
urethane anaesthesia. The exposure of both tubes and
ovaries is had by a simple laparotomy. The ova are held
in a special pipette with an opening in the tube above
the capillary. This type of pipette permits one to take up
a minimum amount of fluid with the eggs, and also prevents
the ova from being drawn into the wide-bored portion of
the pipette. The capillary portion is inserted into the upper
3^ of the tubes and the ova expelled by gentle pressure
METHODS FOR THE MANIPULATION OF OVA 67
on the bulb when the opening in the tube is closed over.
No amount of pressure on the bulb will expel the ova if
the opening is not closed. Extreme care should be taken to
expel only the ova and the fluid containing them. If air is
also pumped into the tubes it often blows the eggs down too
far into the tubes or even into the uterus. Excessive fluid
acts in the same way.
The writer (in collaboration with Dr. E. V. Enzmann)
has also transplanted mouse ova into the fallopian tubes.
Here it is necessary to slit the capsule and expose the tubal
opening, which is slightly wider than at the ampulla, but
not as wide as the rabbit's fimbriated opening. The tubes
are observed under a dissecting microscope and the opening
exposed by manipulation with iris or watchmaker's forceps.
The delicate mouse ova are best handled in warm Ringer-
Locke solution plus serum.
Nicholas (1933a) has transplanted rat ova from the fal-
lopian tubes into the uterus. In this case the tubes are
excised at the isthmus and the ova expelled from a capillary
pipette into the uterine lumen.
CHAPTER VI
THE TUBAL HISTORY OF UNFERTILIZED EGGS
When ovulation occurs without fertilization the liberated
ova enter the Fallopian tubes and eventually degenerate.
In most polyovular mammals the ova are shed surrounded
by an apparently sticky cumulus ovigerus so that a sort of
plug is formed due to the adhesion of the various separate
cumuli (see Plate VI, Figure 1). This cumulus mass remains
more or less intact for some time and then the cumulus cells
gradually become detached so that the ova finally float free.
The opossum (Hartman, 1925) and sheep (Clark, 1934)
appear to be exceptions since very few follicle cells surround
the newly shed ova.
The chronology of egg passage in the tubes is best had
in the rabbit where ovulation occurs at 9 J^ to 10}^ hours
after copulation.
The freshly ovulated ova enter the tubes and become
massed together, due to the adherence of the sticky masses of
cumulus cells. By 11 hours after copulation (about 1 hour
after ovulation) this mass of cumulus cells containing the
ova becomes securely lodged in the narrower portion of the
tubes just below the broad, fimbriated end. On washing
from the uterine end of the tubes this mass (see Figure 1,
Plate VI) is first ejected, then the washing fluid. The ova
remain thus massed together until about 17 hours after
copulation, an occasional ovum separating out of the mass
as early as 16 hours after copulation. Figure 2, Plate VI,
is the photograph of an ovum still embedded in the mass of
follicle cells at 16 hours after copulation. Figure 3 is the
photograph of the single one of the 10 ova removed at the
same time as that of Figure 2 that had separated out of the
mass. Note a number of follicle cells still clinging to the egg.
68
Fig. 1
m^' : m
Fig. 2
Fig.
F"
#
Fig. -4
Fig. 5
Fig. 6
^^r??^S^-'
>r:
-'•:,' ,*
Fig. 7
Fig. 8
Plate VI. (From the Proceedings of the Royal Society.)
Fig. 1, Three ova in the cumulus mass recovered from the fallopian tubes of
rabbit doe 123^ hours after a sterile mating. Fig. 2, An unfertilized ovum still in
the cumulus mass 16 hours after a sterile mating. Fig. 3, Another 16-hour ovum
free of the cumulus mass. Fig. 4, An ovum recovered 19 hours and 5 minutes after
a sterile mating with no adherent follicle cells. Fig. 5, An ovum recovered 243^ hours
after a sterile mating showing a definite albumin coating. Figs. 6-9, All from sterile
matings at the following intervals after sterile copulation: 6. 43 hours, 30 minutes,
7. 73 hours, 40 minutes, 8 and 9. 96 hours, 45 minutes.
69
70 THE EGGS OF MAMMALS
As they separate out of the cuinuhis mass the egjj;s emerge
surrounded more or less by a few adherent folUele cells, and
proceed down the tubes where these few adherent cells are
lost. At 20 hours after copulation all the adherent cells are
gone and a thin layer of albumen is laid down about the
zona pellucida. Eggs washed out at this time show very
clearly the transparent, shining zona pellucida about the
yolky, granular egg cytoplasm, with an extremely thin al-
bumen layer surrounding the zona (see Figure 4). The
process in\'oh'ing the separation of the eggs out of the
cumulus mass and the clearing off of adherent cells
thus involves a period of about 3 hours. When eggs are
washed out during this period one observes in a single
washing all the stages described, eggs completely clear
of adherent cells being preponderant toward the end of
the period. One may even find an occasional egg still
surrounded by adherent cells as late as 20 hours after cop-
ulation.
It is important for reasons that will be obvious later,
to note that by 20 hours after copulation all rabbit ova are
free of follicle cells and have begun to accumulate a layer
of albumen. By 24 hours after copulation this albumen
lajTr is quite appreciable (see Figure 5). Subsequently the
ova descend to the uterine end of the tubes acquiring in their
passage successive layers of albumen so that the albumen
layer may eventually become several times the thickness of
the egg itself (see Figures 6 to 9). The zona pellucida no
longer presents the clear, shining appearance observed before
the deposition of albumen. Most of the ova recovered from
the tubes contain at least one polar body, occasionally two
or even three. In some cases none ha\'e been observed but
this may be ascribed to faulty observation as the eggs often
come to rest with the polar body hidden.
The eggs enter the uterus between 72 and 96 hours after
copulation. No more albumen is added and the eggs undergo
rapid disintegration. It is, in fact, very difficult to recover
unfertilized ova from the uterus. Pincus (1930) was unable
TUBAL HISTORY OF UXFKRTILIZl^D EGGS 71
to obtain the full complement as indicated by the corpora
lutea count. They are either rapidly resorbed or washed
out into the vagina. The cytoplasm of eggs recovered from
the uterus shows distinct evidences of degeneration (Fig-
ures 8 and 9).
The persistence of the corona radiata for some time after
ovulation occurs regularly not only in the rabbit (cj. Yamane,
1930, 1935) but also in the mouse CLong, 1912), the rat
(Gilchrist and Pincus, 1932), the dog fEvans and Cole, 1931),
and man (Allen, Pratt, Newell and Bland, 1930a). It is
notable that opossum ova with no surrounding cumulus
mass enter the uterine portion of the oviduct in approx-
imately twenty-four hours, whereas all available information
indicates that in the higher mammals unfertilized ova enter-
ing the uterus do so at approximately 3 H days after ovula-
tion. In the rat (and probably also the mouse) unfertilized
ova apparently degenerate in the uterine portion of the
tubes (Long and Evans, 1922; Mann, 1924). Albumen
deposition about tubal ova occurs in the rabbit and opos-
sum; in most other mammals the ovum traverses the tube
surrounded only by the zona pellucida.
The dissolution of the cumulus mass surrounding newly
liberated ova seems to involve a definite process in the
tubes and is in all probabiUty not due to an autogenous
change in the cumulus cells themselves. In guinea pigs the
fresh cumulus mass is so tenaciously adherent that it cannot
be completely removed by dissection (Squier, 1932). Gil-
christ and Pincus (1932) found that rat ova incubated in
Ringer's solution did not become free of adherent cells even
after many hours. In rabbit ova grown in blood plasma a
fibroblast-like outgrowth of the cumulus cells occurs but
nonetheless the radial connections to the zona pellucida
are not lost (Pincus, 1930). The writer has also observed a
similar outgrowth from the cells surrounding cultured hu-
man ova, but the extremely tenacious covering of follicle
cells is not lost. The likelihood that a slow enzymatic process
is involved in the freeing of the adherent cells is substanti-
72 THE EGGS OF MAMMALS
ated by the great acceleration of this dehiscence in the
presence of sperm (see Chapter VII).
The unfertihzed ova of most manamals begin to show-
signs of degeneration when they reach the distal portion of
the tubes. In the opossum clear evidences of degeneration
are observed by twenty-four hours after ovulation when the
Fig. 20. Fragmenting opossum egg seven
days after arriving in the uterus. Section of
one of the eggs shown at A, containing three
large chromatin masses almost free of cyto-
plasm. (From the American Journal of Anat-
omy.)
ova enter the uterus (Hartman, 1924). The degenerative
changes have been described in detail by Smith (1925).
The ovum may remain intact but develop a well vacuolized
cytoplasm with clumped or fragmented chromatin. Ordi-
narily, a definite fragmentation of the whole ovum occurs
(Figure 20), and the irregular blastomere-like formations
may contain bits of fragmented chromatin or lack chromatin
entirely. In some 300 opossum ovum sectioned and ex-
amined Smith never observed a true cleavage spindle, and
TUBAL HISTORY OF UNFERTILIZED EGGS 73
appropriately concludes that parthenogenetic development
never occurs. Her statement that pregnant (or psuedo-
pregnant) condition of the
animals should favor par-
thenogenesis is not neces-
sarily correct since activa-
tion may require special
physiological conditions. In
the unmated mouse, how-
ever, Charlton (1917) has
described identical modes
of degeneration in tubal
ova with scarcely an ap-
proach to normal cleavage,
and out of 152 tubal ova
in the unmated rat Mann
(1924) found only three
which appeared to have
undergone a belated parthenogenetic development (see
Table VIII). In the rabbit (Pincus, 1930) fragmentation
occurs rarely; an o\aim of the type shown in Figure 21 is
occasionally encountered.
The fragmenting ova found in the tubes of rats and mice
Fig. 21. Rabbit ovum recovered
from the tubes 411^ hours after ster-
ile copulation showing polar fragmen-
tation. (From the Proceedings of the
Royal Society.)
TABLE VIII
The Conditions of Tubal Ova in Various Portions of the Oviduct
IN THE Rat. (From Mann, 1924)
z
z
z ,
o ,
o
O U
?^
h>-
hS
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as J
^r,
n
^H
g
>
^
P <
^ ss
p £ a
03
s <
o <
^ S!
TD
H a
h O
h OS o
■t a
z a
a
^ 5
a
^
z
< <
< J
< H <
s „
^ 3
S 3
a
?^ ^
a Q
c^
IrH r\
H O
y^
^ ra
'^^ O
■^■^ K
«S
s u
Z P
^
So
J ° «
>J
o >
m
a E 9
lis
ii
o z
m
a z
■< ti ij
a
Oh
< o z
a a a
H O Q
Z OS 2
a a o
PhO
aixas
xnm'A
Ui'J-jU
Um
P^O
c^So
fe
hS
QUO
^
1-3.5
34
4
2
15
2
4-5
6
16
2
1
1
5-8.5
1
7
14
13
1
1
8.5-9
2
6
22
2
Totals
41
4
2
40
16
20
22
2
2
3
74
THE EGGS OF MAMMALS
finally disappear either through complete disintegration or,
what is more hkely, by phagocytosis (Figure 22). They
usually disappear before the succeeding ovulation, although
Hensen (1869) has described the retention in a blocked tube
of about 100 rabbit eggs apparently from several ovulations.
Fig. 22. Section through a fragmented mouse
ovum recovered 81 hours after a sterile mating.
Phagocytes (C) absorb the degenerated cytoplas-
mic particles (E). (From the Biological Bulletin.)
The rate of passage of ova in the tubes and the method
of transport have been the subject of considerable contro-
versy and discussion (see Parker, 1931 and Hartman, 1932&).
It is generally acknowledged that the passage through the
upper portions of the tubes is relatively rapid (Anderson,
1927; Lewis and Wright, 1935) since except shortly after
ovulation both unfertilized and fertilized ova are found for
the most part in the lower two-thirds of the tube. The
method of propulsion of the ova by ciliary and other tubal
movements is adequately discussed by both Parker and
Hartman and will not be entered into here.
CHAPTER VII
FERTILIZATION AND CLEAVAGE
The events occurring at fertilization in the fallopian tubes
have been subject to detailed examination chiefly in poly-
ovular mammals, e.g., the rabbit, rat, mouse, ferret, etc.
In all cases the sperm surround the ova embedded in the
mass of follicle cells, and penetrate to the ova causing the
follicle cells to fall away at the
same time. That the sperm
swarm present in the tubes is
actively responsible for the fall-
ing away of the follicle cell
mass is abundantly evident from
numerous recent observations of
fertilization in the rabbit (Pincus,
1930; Yamane, 1930, 1935;
Pincus and Enzmann, 1932,
1935). As described previously
rabbit ova in does mated to
sterile bucks begin to separate
out of the follicle cell mass by 16
hours after copulation at the earliest, and the process is
normally completed between the 17th and 19th hours.
In fertile matings free ova have been observed as early as
113/^ hours after coitus, and all ova are invariably free
by the 14th hour. Furthermore, when freshly o\ailated
ova from sterile matings are placed in vitro with sperm
suspensions there is a rapid dispersion of the surrounding
follicle cells which does not occur in control cultures of ova
in sperm-free media. Similar phenomena have been observed
by Gilchrist and Pincus (1932) in the rat (Figures 23 to 25)
and by Pincus (unpublished observations) in the mouse.
75
Fig. 23, Rat ovum recov-
ered from the tubes at 16
hours after a sterile mating.
Note surrounding folUcle cells.
(From the Anatomical Record.)
76
THE EGGS OF MAMMALS
Yamane (1930) has ascribed the phenomenon of folHcle
cell dispersion to the presence of a proteolytic enzyme in the
spermatozoa. He was able to secure a similar dispersal of
follicle cells from sperm sus-
pensions heated to 60° C. and
from preparations of pancrea-
tin containing trypsin. Yam-
ane (1930) believes that this
J "^ '^^^^P^^^' J^^H proteolytic enzyme is also re-
f f^&^S^X€jli^S sponsible for the activation
of the egg since he observed
''polar" bodies formed in
rabbit ova exposed to the
suspensions of dead sperm
and to the enzyme prepara-
tions.
Pincus and Enzmann
(1935) have examined this
situation in some detail.
Sperm suspensions free of
seminal fluid were obtained
from the vas deferens of adult
rabbit males. Dilutions were
made with a buffered Ringer-
Locke solution at pH 7.3 —
7.5. The ova were taken
at 12 H to 153^ hours after
copulation from rabbit does
mated to sterile (vasecto-
mized) males ; these ova were
invariably well embedded in
the massed follicle cells. The
procedure followed was to
place the massed ova in the sperm suspension and incubate
for at least two hours. All ova were examined at two hours
after semination and in some instances where no obvious
signs of fertilization were observed incubated for 12 hours.
Fig. 24. Rat ovum of Fig. 23
after 2 hours with Hving
sperm. Note absence of folhcle
cells and protrusion resembling
a polar body. (From the Aiia-
tomical Record.)
Fig. 25. R.it ovum recovered 11
hours after sterile mating and in-
cubated with living sperm for 2
hours. Note shrunken vitellus and
two polar bodies. (From the Ana-
tomical Record.)
FERTILIZATION AND CLEAVAGE
77
In most instances the ova were fixed in Bouin's solution and
sectioned in order to determine the nuclear condition. The
TABLE IX
The Effect of Various Concentrations of Live Sperm upon Freshly
Ovulated Rabbit Ova. (From the Journal of Experimental Zoology)
Concentra-
tion OF
Effect on Cumulus
Date
Sperm per
MM. 3
Cell Mass
Effect on Eggs
6/II/34
(undiluted)
Destroyed in 2 to
2 polar bodies; ova completely
185,000
3 minutes
dissolved after 24 hours
6/II/34
92,500
Destroyed in sev-
eral minutes
2 polar bodies
20/1/34
(undiluted)
Destroyed very
1 polar body after two hours;
90,000
rapidly
polyspermy probable because
of very active sperm suspen-
sion
10/III/34
80,000
Destroyed
2 polyspermic ova, one polar
body; one monospermic with
2 polar bodies
16/1/34
(undiluted)
62,500
2 polar bodies; fertilized
20/1/34
55,000
}f
1 polar body
20/1/34
40,000
"
1 polar body
26/1/34
38,400
}j
2 polar bodies; fertihzed
10/III/34
30,000
Destroyed in 20
1 egg with 3 sperm attached and
minutes
2 polar bodies; 2 eggs with
single sperm attached and 2
polar bodies; not incubated
6/II/34
32,200
Destroyed
2 polar bodies; fertilized
6/II/34
30,000
>)
1 polar body; no sperm entry
26/T/34
25,000
}f
2 polar bodies; fertihzed
26/1/34
14,300
Partly destroyed
1 polar body; not fertilized
26/1/34
10,700
" "
6/II/34
10,100
" "
6/II/34
8,000
n ;>
26/1/34
7,200
)} ft
6/II/34
4,000
" "
26/1/34
3,600
>> }f
2/II/34
6,000
Destroyed almost
1 polar body; \
at once
no fertilization)
2/II/34
3,000
Destroyed in
1 polar body; ( rat
2 minutes
no fertilization/ sperm
2/II/34
1,000
Destroyed in
1 polar body; \
33^ minutes
no fertilization/
sectioned ova of the experiments listed in Table IX invari-
ably showed true polar bodies; no achromatic extrusions
were observed. Furthermore, all ova with two polar bodies
78 THE EGGS OF MAMMALS
contained either attached sperm or male pronuclei, whereas
all ova with single polar bodies showed no signs of sperm
entry with the exception of two heavily polyspermic ova.
Polyspermy may prevent the second polar division, but
probably only when extremely active and dense sperm sus-
pensions are used. The presence of two polar bodies may
therefore ordinarily be taken as a sign of activation.
It is evident from the data of Table IX that both the
degree and speed of dispersion of the follicle cell mass is
roughly proportional to the concentration of the sperm sus-
pensions used and that those sperm concentrations which
fail to effect a complete dispersion of the follicle cell mass
also fail to cause second polar body formation. But rat sperm
as well as rabbit sperm can effect complete dispersal of the
folhcle cells about rabbit ova and yet no polar body forma-
tion occurs. This seems to indicate that the activation of
the o\aim and follicle cell dispersion involve distinct and
separate reactions.
The data of Table X substantiate this conclusion for they
show that sperm-free fluid from the vas deferens and sperm
suspensions heated to 60° C. for a few minutes cause typical
follicle cell dispersion but no polar body formation. That a
heat-labile substance is involved in the follicle cell disper-
sion is evidenced by the data on ova exposed to boiled sperm
suspensions. This substance is probably carried by the
sperm since similar follicle cell dispersion in vivo is brought
about by sperm that have travelled the length of the oviducts.
Yamane (1930) found that both rat and horse spermatozoa
caused second polar body formation in rabbit ova, and since
his pancreatin solutions also caused the same result he con-
cluded that a non-species-specific sperm-borne tryptase was
involved. As shown in Table IX above rat sperm were
ineffective in causing second polar body formation, but they
were more potent than rabbit sperm suspensions in causing
follicle cell dispersion. Accordingly Pincus and Enzmann
(1936a) undertook the experiments with trypsin preparations
presented in Table XL
TABLE X
The Effect of Dead Sperm Preparations and Sperm-Free Seminal
Fluid upon Freshly Ovulated Rabbit Ova.
Experimental Zoology)
(From the Journal of
Treatment of Sperm
Effect on
TTimrr^'T' m>j T^^nnci
Dilution of
Date
Suspensions
Cumulus Mass
Hjr r liiK^x KJj^t xjouo
Preparation
10/1/34
Heated to 60° C.
Destroyed in
1 polar body;
Undiluted
all sperm dead
10 minutes
no fertiliza-
tion
30/III/34
Completely dessi-
Destroyed in
1 polar body
Made up to
cated at room
2 minutes
original vol-
temperature; all
ume
sperm dead
30/III/34
t)
Destroyed in
5 minutes
))
Made up to
original vol-
ume and di-
luted 3^
30/III/34
))
Destroyed in
11 minutes
>f
Made up to
original vol-
ume and di-
luted 34
30/III/34
>»
Destroyed in
21 minutes
>>
Made up to
original vol-
ume and di-
luted Vs
12/1/34
Centrifuged at 3000
R.P.M. for 5 min-
utes; heated to
60° C; superna-
tant fluid used
Destroyed
>j
Undiluted
17/111/34
Centrifuged at 3000
R.P.M. for 40
minutes; heated
to 60° C; super-
natant fluid used
Destroyed in
3 minutes
))
Diluted 1/40
17/III/34
}}
Destroyed in
43/2 minutes
}f
Diluted 1/80
17/111/34
n
Destroyed in
73^2 minutes
Diluted 1/120
17/III/34
j>
Destroyed in
8 minutes
))
Diluted 1/160
12/1/34
Centrifuged at 3000
R.P.M. for 5 min-
Destroyed
3 eggs out of
9 with sec-
Undiluted
utes; not heated;
-
ond polar
supernatant fluid
body and
used; a few sperm
sperm
present
17/III/34
Centrifuged at 3000
R.P.M. for50min-
utes; not heated;
no sperm present
Destroyed in
13^ minutes
1 polar body
Diluted 1/20
20/IX/35
Boiled for 12 min-
utes; all sperm
dead
Left intact af-
ter 1 hour
)>
Diluted H
79
80
THE EGGS OF MAMMALS
TABLE XI
The Effects of Exposing Freshly Ovulated Rabbit Ova to Various
Solutions of Trypsin. (From the Journal of Experimental Zoology)
Trypsin Con-
Date
centration
(Dry Trypsin
PER 100 c.c.
Effect on
Cumulus Cell
.Mass
Effect on Eggs
Ringer-Locke
Solution)
10/11/34
0.50
Destroyed
3 "polar" bodies in 10 minutes
10/11/34
0.25
M
Egg shrunken
10/11/34
0.125
"
>)
10/11/34
0.062
Partly destroyed
M
10/11/34
0.032
»»
"
6/II/34
25.00
Destroyed almost
6 to 10 "polar" bodies followed
immediately
by partial digestion of ova
6/II/34
21.00
"
"
17/11/34
1.00
Destroyed in
1 minute
Egg partly digested
17/11/34
0.50
Destro3^ed in
13^ minutes
1st polar body digested
17/11/34
0.25
Destroyed in
3 minutes
1 polar body, egg shrunken *
17/11/34
0.125
Destroyed in
6 minutes
>) M :tc
17/11/34
0.062
Destroyed in
14 minutes
)) i1 *
17/11/34
0.032
Destroyed in
31 minutes
>> n *
* All these ova showed irregular masses of webbed tissue in the perivitelline space.
The data of these experiments show typical folUcle cell
dispersion and also ^^ polar body" formation (Figure 26).
These are, however, not true polar bodies but rounded cyto-
plasmic masses caused by the action of the enzyme prepara-
tion upon the egg surface. Sections of the ova of these
experiments showed the ''polar bodies" to be chromatin
free. The polar bodies observed by Yamane in his pan-
creatin experiments were probably of this nature. The polar
bodies formed in his experiments with rat and horse sperm
may have also have been false polar bodies due to the
strongly digestive action of the heterologous sperm sus-
pensions, for, as we have seen, rat sperm suspensions are
extremely effective as follicle cell dispersing agents even in
very low concentrations. Krasovskaja (19356) believes that
FERTILIZATION AND CLEAVAGE 81
actual penetration and pronucleus formation occurred in his
attempts to fertilize rabbit eggs with rat sperm. No figures
showing actual sperm penetration are given in this paper.
The nuclear configurations shown may, in fact, occur in
Fig. 26. Rabbit ovum from sterile mat-
ing treated with trypsin solution. Note
many "polar" bodies. See text.
ova cultured in vitro with no sperm added (see Chapter
VIII).
The inamediate effect of semination (and fertilization)
upon mammalian ova is a definite shrinkage of the vitellus
(Pincus and Enzmann, 1932). Quantitative estimates of this
shrinkage in rat eggs have been made by Gilchrist and
Pincus (1932). In Table XII are presented their data on
folhcular and tubal ova. They show that a 14 per cent reduc-
tion in volume occurs in fertilized tubal ova. Furthermore,
when unfertilized ova are exposed to sperm suspensions
a similar shrinkage occurs (Table XIII). This shrink-
age is not due to polar body extrusion since it occurs in vitro
within 5 to 10 minutes, and polar bodies are normally ex-
truded at 45 minutes to 1 hour after semination in vitro
(Long, 1912). The ova apparently increase somewhat in
volume after this initial shrinkage. Krasovskaja (1935a)
82
THE EGGS OF MAMMALS
has observed an exactly similar initial shrinkage followed
by a return to normal in rabbit ova seminated in vitro.
TABLE XII
The Volume of Rat Eggs in Three Stages of Development.
Gilchrist and Pincus, 1932)
(From
Stage
Follicular
Tubal, un-
fertilized
1-cell
Average Volume of
Round Eggs, cu. mm.
0.000333 (1)
0.000251
0.000202
0.000023
0.000009
Average Volume of
Elongated Eggs,
0.000339 ± 0.000017
0.000226
0.000200
0.000013
0.000010
Average Volume of
All Eggs, cu. mm.
0.000337 ^ 0.000010
0.000234
0.000201
0.000018
0.000010
TABLE XIII
The Size of Rat Eggs under Various Conditions of Culture. (From
Gilchrist and Pincus, 1932)
Treatment
Incubated in Ring-
er's solution
alone
Incubated with
live sperm
Incubated with
dead sperm
Num-
ber
OF
Eggs
Average
Diameter
Immediately
AFTER
Putting
Eggs on
Slide,
Microns
74.4 ± 1.4
77.9 ± 1.4
72.8 ± 1.1
Average
Volume
Calcu-
lated,
cu. mm.
0.0002 IG
0.000248
0.000204
Average
Diameter
Some Time
AFTER
Incubation,
Microns
76.3 ± 0.4
72.7 ± 1.4
69.7 ± 1.3
Average
Volume,
Calcu-
lated
cu. mm.
0.000232
0.000205
0.000179
Shrink
age.
Per
Cent
17
12
Sperm penetration into living ova has been observed
only once (Pincus, 1930); a modified fertilization cone ap-
pears to form at the point of contact. This cone very
quickly subsides as is apparent also from fixed preparations
of mammalian ova in the tubes {e.g., Lams and Doorme,
1908; Sobotta and Burkhard, 1911 ; Lams, 1913; and others).
The length of time that the mammalian ovum remains
capable of fertilization has been largely a matter of specu-
lation. Exact experimental inquiry has, however, been
undertaken in the rabbit (Hammond and Marshall, 1925;
Hammond, 1928 and 1934) and in the ferret (Hammond and
Walton, 1934). Taking advantage of the fact that the
FERTILIZATION AND CLEAVAGE
83
Litter Size and Fertility
TABLE XIV
IN Timed Matings of Rabbit Does. (From
Hammond, 1934)
No. OF
Matings
Matings at
Hours
after
Sterile
Coitus
Hours
before ( + )
or after (— )
Ovulation
Average
Litter
Size
Matings
Fertile,
Per Cent
No. OF YOUNQ
PER Mating
Made
(a) All strains together (52 different does used)
323
Normal
+ 10
6.4
79.6
5.3
6
5
+ 5
6.4
82.3
5.3
65
6
+ 4
4.7
64.6
3.0
55
7
+ 3
4.4
58.2
2.5
81
8
+ 2
4.2
42.0
1.8
85
9
+ 1
3.6
37.6
1.4
68
10
4.5
22.1
1.0
57
11
- 1
3.4
12.3
0.4
63
12
- 2
3.2
6.3
0.2
(b) C strain (17 different does used)
131
Normal
+ 10
7.4
75.0
5.0
25
6
+ 4
5.4
52.0
2.8
18
7
+ 3
3.7
55.6
2.1
22
8
+ 2
2.8
27.3
0.8
21
9
+ 1
4.3
28.6
1.2
20
10
4.5
10.0
0.4
18
11
- 1
19
12
_ 2
(c) E strain (21 different does used)
90
Normal
+ 10
8.1
80.0
6.5
3
5
+ 5
7.0
100.0
7.0
19
6
+ 4
5.8
63.2
3.7
23
7
+ 3
5.6
65.2
3.8
37
8
+ 2
4.9
48.6
2.4
48
9
+ 1
3.6
41.7
1.5
25
10
5.4
36.0
2.0
21
11
- 1
4.2
23.8
1.0
21
12
- 2
4.0
9.5
0.4
(d) F strain (14 different does used)
102
Normal
+ 10
4.0
84.3
3.4
3
o
+ 5
5.5
66.6
3.7
21
6
+ 4
3.4
81.0
2.7
14
7
+ 3
2.7
50.0
1.4
22
8
+ 2
3.8
5.5
1.7
16
9
+ 1
2.7
47.5
1.0
23
10
2.2
37.4
0.4
18
11
- 1
1.5
11.1
0.2
23
12
- 2
2.5
18.7
0.2
84
THE EGGS OF MAMMALS
rabbit OMilates at 10 hours after copulation and the ferret
at about 30 hours, Hammond and his coworkers undertook
a series of matings using an initial sterile mating to initiate
the o\ailation stimulus and then fertile mating to permit
sperm access to ova at successively later intervals. In the
80
- 8
- , —
y
70
- 7
_
\
W
\
i-J
..........
......—.-•••, y
1-1
geo
-|6
-
\ \
u
K
'. \
u.
2
___.
- — — > \ \
^50
-h5
_
\ \ \
a
Ed
\ '--A A
H
9
\ "V / \
<40
-^4
—
\ X/ \
S
>
\ X V
Ui
<
O30
- 3
—
\
^
\ \
20
- 2
~
\\
10
- 1
'
s \
\ \
fi
It
I
1 1 1 1 1 1 1 1
+10
+ 5 +4 +3 +2 +1 -1 -2
OVULATION
AVERAGE
UTTER SIZE
^c OF MATINGS
WHICH WERE
FERTILE
NUMBER OF
YOUNG PER
MATING
HOURS INTERVAL BEFORE (I-) OR AFTER (-) OVULATION
Fig. 27. Fertility of matings made at different intervals of time before or
after mating (all strains). (From the Journal of Experimental Biology.)
most extensive series of rabbit matings (Hammond, 1934)
employed three inbred strains of rabbits in order that homo-
geneous conditions of fertility might exist in his experi-
ments. The data of his experiments are given in Table XIV,
and a graphical representation in Figure 27.
It is at once obvious from these data that matings to
fertile bucks made after the 5th hour following a sterile
mating show a decline both in absolute (per cent of fertile
matings) and relative fertihty (number of young produced).
WTien matings are made to fertile bucks at twelve hours
after the sterile copulation, i.e., at two hours after o\ailation
minimum fertility is attained.
FERTILIZATION AND CLEAVAGE
85
In order to make quite certain that the cause of the
smaller litters produced after the experimental matings made
late in relation to ovulation was due to the ova not being
fertilized and not to any interference with the process of
ovulation or other causes, a few does so mated were killed
during pregnancy and the number of corpora lutea {i.e.,
ova shed) compared with the number of foetuses present.
The results are given in Table XV, and demonstrate that
there is a decrease in the number of ova fertilized in the
later matings. This implies that the sperm reach the portion
of the tubes containing the ova at a time when these ova
are for some reason no longer fertilizable.
TABLE XV
The Percentages of Rabbit Ova Fertilized in Matings Made at Vari-
ous Times before and after Ovulation. (From Hammond, 1934)
Matings at
Does
Number of
Ova Not
Hours
before ( + )
or after
(-)
Ovulation
Hours
after
Sterile
Coitas
Number
Strains
Ova
Shed
Normal
Foetuses
Atrophic
Foetuses
Ova Not
Ferti-
lized
LIZED,
Per
Cent
6
7
8
9
11
+ 4
+ 3
+ 2
+ 1
- 1
2
2
3
2
2
E
E,F
E
E
E, F
25
23
41
25
19
15
10
18
6
4
2
3
5
8
7
18
19
15
32
35
44
76
79
On the basis of Heape's (1905) observations that rabbit
sperm reach the tops of the tubes in about 4 hours after
coitus, Hammond concludes that rabbit ova can remain
fertilizable for at most 6 hours after o^Tllation, by allowing
a 2-hour postovulatory interval in the matings made at
12 hours after the ovulation-inducing mating. This period
coincides approximately with the time {i.e., 7 hours) that
it takes for the ova of sterile matings to begin to separate
from the follicle cell mass and start their free travel down
the tubes. Hammond concludes therefore that the presence
of the plug of massed ova is necessary for fertiUzation. He
reasons as follows:
'^The plug, of liquor folliculi and detritus, containing the
ova dams up the top of the Fallopian tube and remains there
86
THE EGGS OF MAMMALS
for some 4 (in fertile matings) to 7 (in infertile matings)
hours, during which time the ascending sperms are collect-
ing in its lower layers (see Figure 28). The accumulation
of sperms so effected ensures that sufficient shall be available
to fertilise the ova as they emerge from the plug. As the
sperms are put in progressively later than normal in relation
to the time of ovulation, the accumulation of sperms be-
comes progressively less and the chances of all the ova
FALLOPIAN TUBE
PLUG CONTAINING OVA
Fig. 28. Diagram illustrating how the chances of the ova becoming fertilized
are reduced as the interval between mating and ovulation is reduced, a =
amount of sperm swarm which would accumulate if mating were made at the
ordinary time — 10 hours before ovulation, b = amount of sperm swarm
which would accumulate if mating were made 4 hours before ovulation. (From
the Journal of Ex-perimental Biology.)
becoming fertilised are reduced in proportion to the time
the fertile mating is delayed with reference to the time of
o\ailation.
^^The ascent of the sperms can be represented as a curve
(see Figure 28 and Hammond and Asdell, 1926) or as a
swarm (in the statistical sense). The apex of the sperm
swarm (shown, in order to assist visualisation of the prob-
lem, very diagrammatically in Figure 28) reaches the top
of the tube just at the time the plug is formed, i.e., at ovu-
lation, and so during the time that the plug exists (about
4 hours) it dams up but few sperms as compared with a
normal mating made 10 hours before ovulation when the
sperm swarm has ascended further (to the point a in Fig-
ure 28).'^
FERTILIZATION AND CLEAVAGE 87
While Hammond's deductions are entirely reasonable, it
is possible that the 6 hours of fertilizable life allotted to
rabbit ova is possibly too short since in normal matings
13^ to 3 hours are required by the sperm to reach the ova.
This would make the critical period some 73^ to 9 hours
long. Furthermore it is not the arrival of the first sperm
that is effective, since as we have previously seen (pages 77
to 78) a definite minimal sperm concentration is necessary
for both folUcle cell dispersion and fertilization. If the
critical period were thereby further lengthened by 1 to
2 hours it would coincide almost exactly with the time
when the ova separating out of the tubal plug begin to ac-
quire a coating of albumen. This coating is impervious to
sperm (Pincus, 1930).
Similar experiments of Hammond and Walton (1934) with
the ferret show that fertile matings made as late as 30 hours
after ovulation result in the production of young. The rea-
sons for the maintenance of the fertilizing capacity of ferret
ova for as long as 30 hours are not deducible in detail since
the exact tubal history of ferret ova is not known. Hammond
and Walton attribute the greater length of fertilizable life
in this case to the longer time it takes for the ova to trav-
erse the oviduct, e.g., 5 to 6 days in. the ferret compared
with 3H days in the rabbit and the presumably correlated
slower dissolution of the plug of massed ova.
In the spontaneously o\ailating mammals the fertilizable
life of the ova is also of short duration, but exact data are
not available since it is ordinarily difficult to ascertain
the specific time of ovulation. Hartman (1924) has shown
that opossum ova traverse the tubal portion of the oviduct
in 24 hours and that upon entry into the uterus unfertilized
ova are definitely degenerated. Charlton (1917) found clear
signs of degeneration in unfertilized tubal mouse ova by
two days after parturition. Since post-partum ovulation
occurs in the mouse at about 14 hours after parturition (Long
and Mark, 1911) mouse ova may be said to retain cyto-
logical normality for about 35 hours. In the rat ova present
88 THE EGGS OF MAMMALS
in the first third of the oviduct appear cytologically normal
(Mann, 1924). According to the data of Long and Evans
(1922) the ova remain in this portion of the oviduct for
about 33 hours. Hartman's (1932a) data on timed matings
in Macacus show that fertile matings occur only between
the 9th and 18th days of the menstrual cycle with maximum
between days 11 and 16. This, of course, does not imply
that the ova are fertilizable for several days, but presumably
that ovulation may occur at any time during the critical
9 day period. Matings time in relation to the onset of
oestrus in the sheep (Quinlan, Mare and Roux, 1932) and
the pig (Lewis, 1911) indicate a maximum period of fertility
of 48 hours.
It is unnecessary in this monograph to discuss the cyto-
logical details of fertilization and cleavage in mammalian
ova, since these are now textbook commonplaces. Our inter-
est is primarily in the physiological mechanisms underlying
these events and their relation to the dynamics of growth
and development. We shall again discuss certain aspects
of the fertilization process in the chapter dealing with the
activation of unfertilized eggs. Now we shall turn our
attention to the relatively scant data that deal with the
mechanism of cleavage in tubal ova.
Until fairly recently no very accurate data on the rate
of cleavage in tubal ova have been available. This has been
due in part to the difficulty of timing ovulation. Even now
it is possible to construct only approximate growth curves
for a limited number of species. These curves are presented
in Figure 29. It will be noted that rabbit ova cleave much
more rapidly than those of the other species (see Plate VII).
It is a matter of some interest to ascertain whether this
difference in the cleavage rate is the result of an especially
stimulating tubal environment in rabbits, or whether the
cleavage rate is an inherent property of the ova. The data
on the monkey were, in fact, deduced from Lewis and
Hartman's (1933) observations of cleavage in vitro, and
may be taken to indicate that segregation from the tubes
FERTILIZATION AND CLEAVAGE 89
results in no great acceleration of cleavage since the growth
rate remains at about the level of the other slow-cleaving
species. The writer has transplanted mouse ova into the
28
20
1 1 1 RABBIT
/ /
++++-t- MONKEY
GUINEA PIG
/ /
MOUSE
■DAT'
f /
f
/
f
/
/
1
KAl
PIG
I
/
/
f
1
•i
1
—
/
1
/ /
/
/A
—
/
J:£:^
J<
^^
'i^^-^^-^'^'^^^^y^
^ ^^5-i
'J^,
..^^
^^^^^S^r
*----
„--'
20 40 60 80
Fig. 29. Showing the cleavage rates of tubal ova in various species of mam-
mals. Abscissa: time in hours after copulation. Ordinate: number of cells.
The rabbit = data of Gregory, 1930, and Pincus, 1930. The monkey = data
of Lewis and Hartman, 1933. The guinea pig = data of Squier, 1932. The
mouse = data of Lewis and Wright, 1935. The rat = data of Gilchrist and
Pincus, 1932. The pig = data of Heuser and Streeter, 1929.
fallopian tubes of the rabbit and has noted no increase in
the cleavage rate over a period of 72 hours.
Castle and Gregory (1929; also Gregory and Castle, 1931)
have, in fact, found certain definite congenital differences in
cleavage rate between different races of rabbits. A resume
of their data is given in Table XVI. The animals of their
large race (A) attain an average adult weight of about
5500 grams in females and 5400 grams in males. The cor-
^
r^-
Mm
Fig. 4
«^"^ ti
Fig. 5
1m y' « ^^
K^
Fig. 6
!^
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13
:^
vf
f':- ^'^
Fig. 14
Fig. 15
)i .^.
#
Fig. 16
Fig. 1-
Fig. 18
Fig. 19
m ♦
^.::-i?^|^ '.T-^^
^#**
♦ 4
Fig. 20
Fig. 21
Plate VII
(Caption on facing page.)
90
Fig. 22
FERTILIZATION AND CLEAVAGE 91
responding adult weights in the small race (B) are 1500 grams
for females and 1400 grams for males. The various hybrid
combinations show roughly intermediate adult weights.
Their data show clearly that certainly beyond the 32nd hour
after copulation the cleavage rate is fastest in the large
race animals and the expected sort of intermediate rates
occurs in the various hybrid combinations. It is entirely
possible that even the earliest cleavages do actually occur
sooner in large race animals since large does ovulate later
than small does and therefore their ova should be fertilized
later. The number of mitoses in cleaving eggs of the large
races also exceeds those in the small race, as the data in the
columns labelled ''prospective" indicate. Since this differ-
ence is consistently present in reciprocal hybrids between
the races the implication is that the sperm nuclei also
participate in the control of the cleavage rate.
In spite of the inherent differences in the speed of segmen-
tation the processes of differentiation occur at the same
time in the large and small size rabbits. Thus the blast o-
Plate VII. All photographs on this plate were made from the living rabbit
eggs in Locke's solution, as soon as possible after removal from oviduct or
uterus at an enlargement of 180 diameters (apochromatic objective 16 mm.,
compensating ocular 8). They are arranged in order of development and
show the principal features of cleavage and formation of segmentation cavity.
It will be noted that the trophoblast is precocious in its differentiation as com-
pared with the remainder of the egg, and as soon as the trophoblast becomes
histologically different one sees fluid begin to accumulate within the egg,
thereby forming the segmentation cavity.
Figs. 4 to 9, Litter C 43, 25 hours after coitus. Fig. 4, one-cell stage with two
polar bodies; Fig. 5, one cell, with coarse granules, perhaps abnormal; Figs. G to
9, showing two primary blastomeres, one tending to be larger than other. Figs. 10
and 11, Litter C 36, 28^:^ hours after coitus. Four-cell stage with crossed arrange-
ment of blastomeres. Figs. 12 to 14, Litter C 45, 32 hours after coitus. 5, 6 and
8-cell stages. In Fig. 13 the cell at top is just dividing. Fig. 15, Litter C 35. 16-cell
stage. Fig. 16, Litter C 41, 55 hours. Morula of about 32 cells. Fig. 17, Litter C 32,
Q9% hours. Smooth surfaced morula. Fig. 18, Litter C 38, 71^ hours. Differen-
tiated trophoblast cells on surface. Fig. 19, Litter C 33, 76^ hours. Fluid beginning
to collect in cleft between trophoblast and inner-cell mass. At this time the albumen
coat is at its maximum. Fig. 20, Litter C 33, 76^ hours. Subtrophoblastic lakelets
of fluid determining early appearance of segmentation cavity. Fig. 21, Litter C 34,
90 hours. Definite segmentation cavity. Note demarcation between trophoblast and
inner-cell mass. Fig. 22, Litter C 42, 92 hours. Zona much stretched and layer of
albumen much thinned out. Inner-cell mass flattening into typical germ-disc.
From Gregory, 1930.
92
THE EGGS OF MAMMALS
TABLE XVI
The Mean Number of Blastomeres per Ovum at Various Times after
Copulation in Large and Small Rabbits and in Certain Hybrids
BETWEEN Them. (From Castle and Gregory, 1929, and Gregory and
Castle, 1931)
Hours
AITEK
Number
Number
Mean Num-
ber OF
Probable
Copula-
Race
OF
Does
OF
Eggs
Blasto-
Error
tion
meres
32M
A (actual)
3
31
4.06
—
>>
A (prospective)
3
31
4.29
—
tf
B (actual and
prospective)
3
12
4.41
—
40
A (actual)
4
45
9.94
±0.24
A (prospective)
4
45
10.82
—
B (actual)
8
27
8.29
±0.19
B (prospective)
8
27
8.37
—
AB (actual)
1
9
8.44
—
AB (prospective)
1
9
8.60
—
41
A (actual)
3
22
11.64
±0.44
A (prospective)
3
22
12.68
—
B (actual)
()
21
8.62
±0.47
B (prospective)
G
21
9.09
—
BD (actual)
2
11
8.63
—
BD (prospective)
B and BD combined
2
11
9.18
—
(actual)
8
32
8.62
—
AD (actual)
3
20
9.25
—
AD (prospective)
3
20
9.55
±0.36
48
F (actual)
4
28
21.75
—
F (prospective)
4
28
22.80
—
B (actual)
4
15
14.00
—
B (prospective)
4
15
14.50
—
A = large race.
B = small race.
AB = Fi hybrid.
BD = seven-eights small (D = AB XB).
F = three-quarters large (AB X A).
actual = number of blastomeres observed.
prospective = number of blastomeres observed plus the number of mitoses.
dermic vesicle forms at the end of the 3d day (Plate VII,
Figs. 18-20), and the embryonic disc by the 168th hour
after coitus. Castle and Gregory therefore attribute large
size to an inherent mitotic intensity independent of dif-
ferentiation potentials.
The ova of the rabbit begin their differentiation early
in comparison with the eggs of other species. Thus Gregory
(1930) detected the beginning of the formation of the inner
cell mass just after the 16-cell stage at about 47 hours after
FERTILIZATION AND CLEAVAGE 93
coitus (37 hours after ovulation) and the cavity of the
blastodermic vesicle may begin to form while the ova are
still in the tubes. Guinea pig (Squier, 1932) ova enter the
uterus in the 8-cell stage at the end of the 3d day after
copulation and the blastodermic vesicles form only in the
uterus at about 43^2 days after coitus. In the rat (Huber,
1915) the ova enter the uterus during the 4th day after
coitus in about 12 cells and start to form the blastodermic
vesicle during the 4th to 5th days post coitum, and in the
mouse (Enzmann, Saphir and Pincus, 1932; Lewis and
Wright, 1935) blastocyst formation occurs in the uterus
during the 4th day after copulation.
The physiological factors governing the cleavage of mam-
malian ova have been scarcely examined. It has already
been stated that the whole course of cleavage of rabbit
eggs may proceed normally in vitro and in heterologous as
well as homologous blood plasma (Pincus, 1930). This
would seem to imply that no special environmental factors
supervene in the tubes. On the other hand the ova of mice,
rats and guinea pigs do not cleave under the ordinary
(or a variety of) tissue culture conditions. The reasons for
this species difference are not known though the superior
vitality of rabbit ova has been attributed to their unique
albumen coating; but Lewis and Hartman (1933) have over
a period of approximately 24 hours, observed the regular
cleavage in vitro of a monkey o\aim which lacks an albumen
coating.
In the case of those ova which have not undergone cleav-
age in vitro one can only deduce that some limiting factor
obtaining in vivo has not been duplicated. Since it is known
that the secretory activity of the tubal epithelium is under
hormonal control of the ovary (c/. Snyder, 1923) it is pos-
sible that a special contribution to the economy of cleaving
ova is made by a hormonally induced secretion. The cleaving
ova of all mammals journey through the tubes during the
early life of the corpus luteum. The secretory activity of
the tubal epithelium changes markedly during the transi-
94 THE EGGS OF MAMMALS
tion from the oestral to the luteal phase. Furthermore, it
is possible that the ovarian hormones themselves may di-
rectly affect the cleavage process. Oestrin, for example,
definitely stimulates the mitotic activity of the vaginal
epithelium, progestin inhibits uterine mitoses, etc.
Accordingly Burdick and Pincus (1935; also Pincus and
Kirsch, 1936) have investigated the effect of ovarian hor-
mones upon the development of rabbit and mouse ova.
They found that the injection of large amounts of oestrin
in no way affected the cleavage process although ova in the
early uterine stages degenerate and die when only moderate
amounts of this hormone are injected (see Tables XXIII
to XXV, pages 118-120, 122). That the hormone injected
definitely affected the tubal tissue was evidenced by the fact
that in both mice and rabbits an effective closure of the
tubo-uterine junction was attained, and in rabbits both
the contractile activity and the histological appearance of
the tubal tissue were definitely altered to the oestrus type.
In addition (Pincus and Kirsch, 1936) it was found that
rabbit ova grow^n in cultures containing appreciable amounts
of oestrin continued to cleave at the normal rate. Finally
fertilized rabbit ova in 1- and 2-cell stages were injected
into the fallopian tubes of does on heat (and therefore
lacking corpora lutea), and these were found to develop
normally up to the early blastocyst stage. Corner (1928)
had already shown that in bilaterally ovariectomized rabbit
does egg development stops at the early blastocyst stage.
The segmentation processes appear, therefore, to be inde-
pendent of the secretory activity of the ovaries, and of any
effect that the ovarian condition may have upon tubal
secretion. Rabbit ova will, indeed, go through the morula
stage in a carefully balanced buffered Ringer-Locke solu-
tion, indicating a fairly complete lack of dependence upon
any special organic nutrition. It has, of course, been re-
peatedly noted by observers of living material {e.g., van Ben-
eden, 1875; Gregory, 1930; Gilchrist and Pincus, 1932;
Squier, 1932) and by those who have examined fixed speci-
FERTILIZATION AND CLEAVAGE 95
mens (Sobotta, 1895; Huber, 1915; and others) that mam-
malian ova show no appreciable increase in size until the
blastocyst stage.
The most convenient approach to the study of the physio-
logical processes underlying segmentation has involved the
study of the respiratory processes (Warburg, 1908-14 ; J. Loeb
and Wasteneys, 1912-15; J. Loeb, 1913; Runnstrom, 1930;
Whitaker, 1933; and others). Mammalian ova are available
in such small numbers that exact quantitative measurements
of respiratory activity are difficult to make and have not
been made. Nonetheless some indication of the nature of
the underlying processes may be had by the use of specific
poisons known to combine with and inhibit the reactions
of definite components of the chain of reactions involved in
respiration. Thus HCN is known to combine with iron-
containing enzyme phaeohemin which is the initial activator
in the aerobic phaeohemin-cytochrome chain (Warburg,
1932) and so to inhibit the respiration involving phaeo-
hemin activity. Cyanide also inhibits the cleavage of ova of
non-mammalian forms (Lyon, 1902; J. Loeb, 1906; see
Needham, 1932), as does an oxygen-free medium (J. Loeb,
1895). Runnstrom (1935) has demonstrated that the mitotic
process at segmentation in sea-urchin eggs is not dependent
upon the level of respiration since the addition of pyocyanine
to cyanide-inhibited egg suspensions restored oxygen con-
sumption to normal levels but no division ensued.
Rabbit ova presumably develop in a medium relatively
low in oxygen, since the oxygen tension of the abdominal
cavity, and by inference that of the tubes (which have free
access to abdominal fluids), is 40 mni. Hg (Campbell, 1924)
as compared with 150 mm. Hg, the oxygen tension of the
air. It is of interest to inquire whether the segmentation of
rabbit ova is Hnked with the aerobic phaeohemin system.
Pincus and Enzmann (19366) have added KCN in appropri-
ate concentration to cultures of cleaving rabbit eggs and the
segmentation has ceased. Cinematographs of these ova
indicated that the eggs were not ''killed" by the poison
96 THE EGGS OF MAMMALS
since they exhibited the cyclosis (cytoplasmic movements)
typical of living ova. Similar experiments with iodoacet-
amide added to the cultures showed normal cytoplasmic
activity of the ova but a limited amount of cleavage. lodo-
acetamide presumably combines with the coenzyme con-
cerned in the reduction of pyruvic to lactic acid (Meyerhof
and Kiesling, 1933) so that the inhibition of both the oxygen-
activating system and its presumable substrate system re-
sults in the arrest of cleavage. While the exact coupling of
the respiratory system with the mitotic mechanism has
yet to be delineated these data do demonstrate that the
fundamental processes are aUke in manomalian and non-
mammahan ova.
We have seen that rabbit ova may be fertilized and cul-
tured in vitro. It is a matter of some importance' to deter-
mine whether such ova may give rise to normal rabbits.
Accordingly the writer (see Pincus and Enzmann, 1934)
undertook the transplantation of such ova into the oviducts
of pseudopregnant rabbit does and found that ova fertilized
in vitro and also normally fertilized ova kept in culture during
the cleavage period apparently resumed normal development
after transplantation as evidenced by the production of
normal young at term. It is a matter of some interest to
note that one set of ova had failed to cleave during 20 hours
in culture but nonetheless young were obtained.
The development of a technique for the transplantation
of mammalian ova into the oviducts makes possible the
testing of a number of problems of development hitherto
inaccessible. As we shall see later (Chapter IX) it is neces-
sary that a progestational uterus be available for ensuring
differentiation of uterine stages. Thus Biedl, Peters and
Hof stater (1922) transplanted rabbit ova into non-pregnant
uteri in some 70 experiments and in only one doubtful case
were young recovered. Nicholas (19336) transplanted the
isolated blastomeres of the 2-cell stage in the rat under the
kidney capsule and observed varying degrees of development
of the three germ layers and their various derivatives. The
FERTILIZATION AND CLEAVAGE 97
writer has transplanted single blastomeres of 2-cell rabbit
embryos into the tubes and obtained normally differentiat-
ing, but small sized blastodermic vesicles from the pseudo-
pregnant uteri of the recipient does. The physiological
processes occurring in such embryos are of extraordinary
interest and certainly deserve further investigation.
CHAPTER VIII
THE ACTIVATION OF UNFERTILIZED EGGS
We have seen that the fundamental control of the cleavage
mitoses is alike in rabbit and sea-urchin ova. We shall now
inquire whether the activation of mammalian eggs is also
similar to that of other forms.
With the exception of the three 2-cell rat eggs described
by Mann (1924) there are no observations of a possible
normal parthenogenetic development of unfertilized tubal
eggs in vivo. With the exception of a single observation by
Champy (1927), the first investigation of the behavior of
unfertilized tubal ova placed in tissue culture is that of
Pincus (1930). His data are presented in Table XVII.
TABLE XVII
The
Development
OF Unfertilized Rabbit Ova in Culture.
(From
Pincus
, 1930)
Age of Ova
(Hours
AFTER Cop-
Num-
ber
Medium
Exam-
ined
(Hours
in Cul-
Description
Num-
ber Di-
vided
Num-
ber
Undi-
ulation)
ture)
(1)
1
RPRE
44
1 — unsegmented
1
(2)
4
RPCE
48
3 — unsegmented
1 — in 3 regular cells
1
3
(3)
4
RPCE
48
4— unsegmented
4
(4)
11 9
1
CPCE
48
1 — several polar
bodies (?)
1 CO
(?)
(5)
11 25
5
RPRE
24
3 — unsegmented
1 — 8 regular cells
1 — 4 regular cells
2
3
(6)
11 40
2
RPRE
48
2 — unsegmented
2
(7)
12 5
5
RPRE
47
3 — unsegmented
2— in 12 to 16 regular
cells
2
3
(8)
12 30
6
RCPCE
27
1 — in 2 regular cells
1 — in 3 regular cells
1 — in 4 regular cells
3 — in 5 to 6 regular
cells
6
(C = Chicken.
R = Rabbit.
P = Plasma.
E = Embryo Extract.)
THE ACTIVATION OF UNFERTILIZED EGGS 99
TABLE XVII (Continued)
The Development of Unfertilized Rabbit Ova in Culture. (From
Pincus, 1930)
Age of Ova
(Hours
AFTER Cop-
ulation)
13 15
13 35
(11)
(12)
13
14
50
(13)
14
35
(14)
15
(15)
15
15
(16)
16
(17)
17
10
(18)
17
33
(19)
17
45
Num-
ber
Medium
RPCE
RPRE
RPRE
RPRE
RPCE
CPCE
RPRE
RPCE
RPCE
CPCE
RPCE
Exam-
ined
(Hours
IN Cul-
ture)
47
43
25
27
30
27
22
23
48
26
Description
2 — unsegmented
1—16 to 20 cells
1 — in 2 cells and 2 to
5 polar bodies
3 — with about 5 polar
bodies
7 — unsegmented
2 — unsegmented
1 — in 4 regular cells
and 2 polar bodies
1 — in morula
1 — unsegmented
2 — in 3 regular cells
2 — in 4 regular cells
2— in 36 to 40 regular
cells
2 — in about 4 cells
regular
1 — with multiple polar
bodies
1 — unsegmented
1 — in 4 regular cells
1 — in about 16 cells
2 — in 1 very large cell
and 10 to 12 small
ones
2 — in about 16 cells
1— in 32 to 48 cells
1 — in 2 regular cells
and 2 polar bodies
1 — in 3 to 4 large
cells and 10 small
cells
1 — in 1 large cell and
16 small cells
2 — no segmentation
1 — 2 large, 2 small
cells and several
polar bodies
1 — in 16 very regular
cells
2— in 20 to 32 cells
Num-
ber Di-
vided
Num-
ber
Undi-
vided
(C = Chicken.
R = Rabbit.
P = Plasma.
E = Embryo Extract.)
100
THE EGGS OF MAMMALS
TABLE XVII (Continued)
The Development of Unfertilized Rabbit Ova in Culture.
Pincus, 1930)
(From
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
Age of Ova
(Hours
AFTER Cop-
ulation
18 10
18 li
18 20
18 25
18 30
18 50
19 5
19 30
19 45
Num-
ber
Medium
RPCE
RPCE
CPCE
RPCE
RPRE
RPRE
RPCE
RPCE
RPCE
Exam-
ined
(Hours
IN Cul-
ture)
48
29
47
24
48
47
22
27
22
Description
3 — unsegmented
1 — 2 unequal cells and
7 to 8 polar bodies
1 — 3 cells and several
polar bodies
1 — 4 regular cells
1 — 10 small cells and
1 large cell
4 — unsegmented
1 — 3 cells regular
2 — about 4 regular
cells, but shrunken
1 — in 12 regular cells
1 — in about 8 cells,
but shrunken
1 — unsegmented
1 — 3 polar bodies
2 — unsegmented
1 — 1 large cell and 2
to 3 small cells
1 — 2 regular cells and
2 polar bodies
1 — 4 regular cells
1 — 7 regular cells
4 — about 8 cells
2 — unsegmented
1 — in 3 cells
2 — in 8 regular cells
1 — in 10 regular cells
2 — unsegmented
2 — in 2 regular cells
4 — in 4 regular cells
1 — in 7 cells
1 — 2 unequal cells and
3 polar bodies
1 — unsegmented
1 — 2 regular cells
2 — 4 regular cells
1 — unsegmented
3— in 2 cells
2 — in 4 cells
1 — in 6 cells
1 — in 8 cells
Num-
ber Di
VIDEO
1(?)
8
Num-
ber
Undi-
vided
(C = Chicken
R = Rabbit.
P = Plasma.
E = Embryo Extract.)
THE ACTIVATION OF UNFERTILIZED EGGS 101
The Development of
TABLE XVII (Continued)
Unfertilized Rabbit Ova
Pincus, 1930)
IN Culture. (From
Age of Ova
Exam-
Num-
(Hours
after Cop-
Num-
ber
Medium
ined
(Hours
IN Cul-
Description
Num-
ber Di-
vided
ber
Undi-
ulation )
ture)
vided
(29)
20
2
CPCE
22
2 — many polar bodies
2
(30)
20
10
3
RPRE
49
3 — in many cells
3
(31)
20
20
4
RPCE
47
4 — unsegmented
4
(32)
20
20
2
CPCE
48
1 — unsegmented
1—1 large and 2 to 3
small cells
1
1
(33)
24
25
5
RPCE
27
1 — 2 unequal cells
3—2 regular cells
1 — 3 regular cells
5
(34)
24
45
2
RPRE
44
1 — 1 large cell and sev-
eral polar bodies
1—16 to 20 regular
cells and a few po-
lar bodies
2
(35)
27
35
5
CPCE
46
1 — unsegmented
2 — about 8 cells and
many polar bod-
ies
2 — one large cell and
many polar bodies
4
1
(36)
28
35
9
RPCE
47
4 — unsegment ed
1 — 4 regular cells
1 — 6 unequal cells
3— about 8 cells
5
4
(37)
37
20
2
RCPCE
27
2 — in many cells and
degenerate
2
(38)
40
40
3
CPCE
45
3 — in many small cells
3
(39)
43
10
6
CPCE
46
1— in 2 cells
1 — in 4 cells and many
polar bodies
2 — in 1 cell and many
polar bodies
2 — in many small cells
6
(40)
47
30
6
RPCE
22
3 — unsegmented
1 — in 2 unequal cells
2 — with many polar
bodies
3
3
(41)
48
30
8
RPCE
48
5 — unsegmented
3 — with many polar
bodies
3
5
(42)
48
47
6
RPRE
52
1 — about 8 large cells
1 — 3 unequal cells and
many polar bodies
6
(C = Chicken.
R = Rabbit.
P = Plasma.
E = Embryo Extract.)
102
THE EGGS OF MAMMALS
TABLE XVII (Continued)
The Development of Unfertilized Rabbit Ova in Culture. (From
Pincus, 1930)
Age of Ova
(Hours
AFTER Cop-
ulation)
Num-
ber
Medium
Exam-
ined
(Hours
in Cul-
ture)
Description
Num-
ber Di-
vided
Num-
ber
Undi-
vided
4— with many polar
bodies
(43)
(44)
(45)
50 30
68 33
72
5
3
4
RPRE
RPRE
RPCE
45
45
22
1 — unsegmented
4 — in many cells
3— unsegmented and
degenerate
2 — unsegmented and
shrunken
2 — about 10 polar bod-
ies and shrunken
4
2
1
3
2
(46)
73 40
7
RPCE
45
4 — unsegmented
2—16 regular (?) cells
1 — 5 cells and 3 polar
bodies
3
4
(47)
96 45
2
RPCE
46
2— unsegmented
2
(C = Chicken.
R = Rabbit.
Plasma.
E = Embryo Extract.)
The primary and surprising fact evident from the data
is that a majority of the ova placed in culture underwent a
certain degree of development, so that out of 213 eggs cul-
tured, 136 or 63.8 per cent are classified as having ^^ divided,"
the term ^'divided" including any degree of observable
development beyond the 1 -celled state of the ova as re-
covered from the animals. It was the primary objective
of these investigations to ascertain the nature of the various
degrees of development undergone in vitro and to establish
any relationship that might exist between the age of the
ova and the nature of the development. Before undertaking
any detailed analysis of the data it is deemed advisable to
describe the various types of development observed.
The ova observed in the 2-cell stage varied in appear-
ance as shown in Plate VIII, Figs. 1-3. The great major-
ity of them resembled that of Figure 1, and showed usually
one, sometimes two or three, polar bodies. The ovum of
Figure 3 was photographed after the egg had been in cul-
f
Fig. 1
Fig. 2
Fig. 3
Fig. 4
»
Fig. 5
Fig. 6
Fig. 7
Fk.. S
Fig. 9
Fig. 10
Fig. 11
Fig. 12 Fig. 13
Fig. 14
L«, A
Fig. 15
Plate VIII. Ova from sterile matings as they appeared after being cul-
tured in vitro. (From the Proceedings of the Royal Society.)
Recovered at 18 hrs.
Recovered at 27 hrs.
Recovered at 19 hrs.
Recovered at 18 hrs.
Recovered at 18 hrs.
Recovered at 19 hrs.
Fig. 7, Recovered at 19 hrs.
Fig. 8, Recovered at 28 hrs.
Fig. 9, Recovered at 17 hrs.
Fig. 10, Recovered at 24 hrs.
Fig. 1,
Fig. 2,
Fig. 3,
Fig. 4,
Fig. 5,
Fig. 6,
30 mins.
30 mins.
5 mins.
10 mins,
15 mins
5 mins.
5 mins.
35 mins.
10 mins,
45 mins. after sterile copulation cultured for
Fig. 11, Recovered at 37 hrs. after sterile copulation cultured for 6 hrs. Fig.
covered at 73 hrs. 40 mins. after sterile copulation cultured for 45 hrs. Fig.
covered at 73 hrs. 40 mins. after sterile copulation cultured for 45 hrs. Fig.
covered from the ovary, and cultured for 28 hrs. Fig. 15, Recovered at
30 mins. after sterile copulation cultured for 24 hrs.
after sterile copulation cultured for
after sterile copulation cultured for
after sterile copulation cultured for
after sterile copulation cultured for
after sterile copulation cultured for
after sterile copulation cultured for
^fter sterile copulation cultured for
after sterile copulation cultured for
after sterile copulation cultured for
44 hrs.
25 hrs.
22 hrs.
17 hrs.
28 hrs.
22 hrs.
22 hrs.
23 hrs.
23 hrs.
24 hrs.
12, Re-
13, Re-
14, Re-
48 hrs.
103
104 THE EGGS OF MAMMALS
ture 22 hours. It was subsequently replaced, and when
examined 24 hours later had formed eight cells quite regular
in appearance. Note is made of this fact because it indicates
that ova segmenting irregularly at the first division may
eventually assume an appearance characteristic of ova under-
going quite regular division. The ovum of Figure 4 was
photographed just as segmentation from two to three cells
was being completed. One of the two blastomeres had not
quite rounded out at the time of photographing. The
segmented ovum of Figure 5 is also in three cells. When
first examined after 23 hours of culturing no segmentation
had occurred; 5 hours later the ovum had divided as photo-
graphed. The ova of Figure 5 were recovered at 18 hours
and 15 minutes after copulation and were still surrounded
by a number of follicle cells. They were placed vis-a-vis
in culture and the out-growing follicle cells of each ovum
became intermingled and caused the compression of the ova
seen in the photograph. Figure 6 is a photograph of a typical
4-celled stage, exactly comparable to the 4-celled stage
of fertilized ova (see Plate VII, Figs. 10 and 11). The num-
ber of polar bodies in such ova vary from one to three.
Again, the great majority of ova observed in four cells pre-
sented the regular appearance of the ovum of Figure 6.
Figure 7 represents an ovum containing seven cells in which
one of the four blastomeres of the 4-celled stage divided
twice while the others remained quiescent. Such differential
division may begin after the 2-celled stage as illustrated
by Figure 8, in which one of the original two cells has re-
mained quiescent while the other divided in two, and one
of the two cells formed divided twice to form four small
cells. There is also photographed the single polar body of
this ovum. Figure 9 represents another case in which one
of the early blastomeres has remained quiescent while the
others have gone on dividing at a rapid rate. Some such
process is responsible for most of the irregular segmentations
observed. At the same time segmentation may proceed in a
manner comparable to that of normal fertilized ova in vivo,
THE ACTIVATION OF UNFERTILIZED EGGS 105
so that one may observe in the same culture the different
types described. Figure 10 is a photograph of an ovum
segmented to about 20 cells and apparently with a marked
degree of regularity. When we come to consider ova seg-
mented into 20 and more cells the interpretation of the
course of their development becomes difficult because of a
peculiar complication. The o\aim of Figure 11 offers a per-
tinent illustration. It w^as recovered from the tubes at
37 hours after copulation and was in the 1-cell stage.
Six hours later it presented the appearance shown in the
photograph. It has apparently segmented into about 36 cells
in the course of 6 hours. This means astonishingly rapid
segmentation. As a matter of fact what probably occurred
was a complex fragmentation of the entire ovum. In the
course of filming an ovum recovered at 29 hours and 20 min-
utes after copulation the course of such fragmentation was
observed. After an initial period of quiescence the ovum
underwent a period of activity which resulted in the sudden
appearance of many small ^^ blast omeres." This was fol-
lowed by a complete quiescence with the cessation of all
cytoplasmic movements. The ''cells" of this fragmented
ovum, however, were not at all distinct in form or outline.
One may observe ''many-celled" ova. in culture that pre-
sented this vagueness of cell outline, but we have also seen
well advanced ova in which the component blastomeres were
as distinct and clear as in the normal fertilized ovum. Inter-
pretation must, therefore, proceed slowly until the exact
mechanics of division in vitro is thoroughly investigated. A
certain amount of light, however, is shed on the problem by
the consideration given below to the relation between the
age of the ova and the nature of the development observed.
Figures 12 and 13 are photographs of two ova recovered at
73 hours and 40 minutes after copulation. They were
photographed after having been 45 hours in the same cul-
ture. Note the remarkable regularity of the cells of the ovum
of Figure 13. The ova of Figures 14 and 15 represent types
ordinarily described as "with many polar bodies." Both
106
THE EGGS OF MAMMALS
have a very large single cell, beside which lie a number of
very small ''cells" comparable in appearance to polar bodies.
Very often this group of ''polar bodies" resembles an ir-
regular indented cytoplasmic mass, and I have actually seen
it formed as such a mass budded or divided off from the
main body of the cell. This represents the extreme of
irregularity observed.
The foregoing account has been given irrespective of the
age of the ova figured. It remains for us to ascertain if any
relation does exist between the age of the ova cultured and
the nature of their development. Before proceeding to a
detailed inquiry, however, it must be pointed out that the
various types of ova described and figured in the photo-
graphs have been observed in ova of all ages so that no
absolute correlation exists. Ova have been considered as
segmenting regularly only when the cells of the two, four,
eight and sixteen cell stages have been of equal size, or
when one could obviously trace the regular descent of the
cells in ova exhibiting intermediate stages. In the cases of
ova exhibiting many cells only those showing clear cell
outlines and cells of equal size have been classified as
"regular."
TABLE XVIII
Effect of Age of Ova when Removed from Doe on Subsequent
Regularity of Division in Vitro. (From Pincus, 1930)
Group
Number
Age of Ova (Hours after
Copulation)
Regular
Irregular
Percentage
Regular
(1)
(2)
(3)
11 to 17
17 to 21
24 to 96
All ova
26
37
10(?)
73
8
16
26
50
76.4
69.8
27.7
59.3
In Table XVIII the data are collected into three groups
as follows: (1) Ova recovered when practically all were in
the cumulus mass; (2) ova separating out of cumulus mass
and not yet covered with albumen ; (3) ova covered with the
albumen deposit. It is obvious from these data that the
THE ACTIVATION OF UNFERTILIZED EGGS 107
percentage of ova segmenting with any semblance of reg-
ularity decreased perceptibly with the age of the ova. In
the group of ova recovered at 24 to 96 hours after copulation
16 of the ova classified as irregular exhibited one large cell
and ''many polar bodies." In fact, 23 or about half of all
the ova called ''irregular" are of this type. A number of
ova, particularly in the 24 to 96 hour group exhibited "many
polar bodies" and a varying number of larger cells. The
rest of the ova classified as irregular were either "many-
celled" with indistinct cell outlines, or contained cells of
unequal size traceable, probably, to the differential division
of early blastomeres.
Now this fact that the younger ova tend to segment
regularly is presumably related to the state of the egg cyto-
plasm. The older ova undoubtedly undergo a certain degree
of degeneration as they progress down the tubes, and the
degree of cytoplasmic degeneration is probably related to
the regularity of the subsequent development in culture.
The problem is unfortunately complicated by the fact that
all ova in culture stop segmenting and degenerate after
some time. In these experiments it is probable that prac-
tically no development occurs after the ova have been in
culture for 36 hours. The time in which the ova may exhibit
their potentialities for parthenogenetic development is, un-
der the conditions of these experiments, therefore extremely
limited. The surprising fact is that such a large proportion
of the ova do exhibit a degree of development that must be
classified as parthenogenetic.
The morphology and cytology of parthenogenetic ova
have been studied in a number of invertebrate forms where
parthenogenetic development has been induced by various
methods of treatment. In almost all cases a very large
proportion of the parthenogenetic ova exhibit marked ir-
regularities in development {e.g., Wilson, 1901; Scott, 1906;
Morris, 1917). In fact all the irregular types described here
have been observed in artificially parthenogenetic inverte-
brate ova. The proportion of regular divisions observed
108 THE EGGS OF MAMMALS
in these ova compares favorably with those observed in
invertebrate ova, with the possible exception of the sea-
urchin eggs, a very large proportion of which (as much as
100 per cent) may develop regularly into swinaming larvae
(Hindle, 1910; Loeb, 1913).
It was not possible to make any extensive cytological
study of the ova described. The few sectioned and stained
eggs obtained, indicate that in ova segmenting regularly
the nuclei and cytoplasm are normal in appearance. In
ova segmenting irregularly the situation is apparently rather
comphcated. There are obvious evidences of degeneration.
Some cells contain nuclei, others do not, and the cytoplasm
is often quite degenerate. One observes ova with several
nuclei and no distinct cell divisions. In the case of one fairly
regular ovum there were at least 37 chromosomes in an
incomplete metaphase plate.
Upon consideration of the various factors involved in
the technique of explanting the ova it seemed most likely
that those young ova which underwent a normal partheno-
genetic cleavage were stimulated by a gradually developed
hypertonicity of the culture medium. For in these experi-
ments the ova were cultured in watch glasses in a moist
chamber, where the evaporation of a small amount of water
from the plasma culture was possible. If this conclusion
is true then at least one of the many types of parthenogenetic
stimuli known to be effective with non-mammalian ova is
similarly stimulating to mammalian eggs.
In order to examine this question further the writer and
Dr. E. V. Enzmann (Pincus and Enzmann, 1936a) have
studied the effect of known methods of parthenogenetic
stimulation upon rabbit ova. We took as our criterion of
activation the production of the second polar body, which,
as we have seen in the experiments with semination in vitro,
is entirely adequate.
The data of these experiments are given in Table XIX.
They demonstrate that short treatment with solutions of
relatively low hypertonicity are certainly effective in in-
THE ACTIVATION OF UNFERTILIZED EGGS 109
TABLE XIX
The Effect of Various Treatments upon the Activation of Rabbit
Ova IX Vitro. (From the Journal of Experimental Zoology)
Date
18/1/34
24/1/34
24/1/34
24/1/34
20/IX/35
20/IX/35
21/IX/35
21/IX/35
21/IX/35
20/IX/35
20/IX/35
21/IX/35
21/IX/35
18/IX/35
Treatment
3 minutes in 2.8 c.c. H/10 butyric
acid + 50 c.c. Ringer-Locke fol-
lowed by 3 mins. in 8 c.c. 2.5%
NaCl -f 50 c.c. Ringer-Locke
followed by plasma culture
3 minutes in 5 c.c. N/10 butyric
acid -f 100 c.c. Ringer-Locke
followed by hypertonic solution
as above
3 minutes in 7.5 c.c. N/10 butyric
acid -|- 100 c.c. Ringer-Locke
followed by hypertonic solution
as above
3 minutes in 10 c.c. N/10 butyric
acid -]- 100 c.c. Ringer-Locke
followed by hypertonic solution
as above
10 minutes in 1.8% Ringer-Locke
5 minutes in 1.8% Ringer-Locke
8 minutes in 1.8% Ringer-Locke
8 minutes in 1.6% Ringer-Locke
8 minutes in 2.0% Ringer-Locke
2 minutes exposure to 45.5° C.
3 minutes exposure to 45.5° C.
2^ minutes exposure to 45.5° C.
3 minutes exposure to 45.5° C.
2 minutes exposure to 60° C.
Result
Cumulus partly dispersed ; one
ovum with 2 polar bodies;
7 with 1 polar body; much
shrinkage
Cumulus partly dispersed; 2
ova with 2 polar bodies;
with 1 polar body; much
shrinkage
Plasmolysis of ova
Plasmolysis of ova
Cumulus intact; only 1st polar
body
Cumulus intact; only 1st polar
body
3 polar bodies in 5 hours
1 egg with 2 polar bodies;
1 egg with 3 polar bodies
2 polar bodies in 3 hours
3^ with 2 polar bodies
2 or 3 polar bodies per egg
2 polar bodies formed
2 or 3 polar bodies per egg
No polar body formation
ducing activation, and that more drastic treatment (e.g.,
longer treatment, or Loeb's treatment) is only occasionally
effective. This indicates that the optimum conditions for
the activation of rabbit ova are different from those em-
ployed with sea-urchin eggs. - The data on the experiments
with ova heated to 45° to 47° show that this heat treatment
is most effectively activating.
We may conclude therefore that certain of the methods
ordinarily employed in the artificial activation of non-
mammalian ova are also effective in activating mammalian
eggs. In a preliminary group of experiments (unpublished
110 THE EGGS OF MAMMALS
data) the writer has transplanted ova so activated into the
fallopian tubes of pseudopregnant rabbit does and has later
recovered the transplanted ova. A number had undergone
normal but obviously belated cleavage. A few cleaved at
the normal rate and about 10% of the total attained the
blastula stage.
In order to obviate any undetected effects of the manipula-
tion of ova in vitro Pincus and Enzmann (1936a) undertook
the activation of ova in vivo by injecting into the tops of
rabbit fallopian tubes sperm suspensions previously irradi-
ated with ultraviolet light of 2357 A° wavelength. The does
used in these experiments had been mated to sterile bucks
12 to 13 hours previously so that their ovulated ova were
embedded in the follicle cell plug. Into one oviduct the
rayed sperm were injected, into the other an identical
sample of unrayed sperm. It was found that ova from the
tubes receiving unrayed sperm suspensions were for the
most part normally fertilized and cleaved at the normal
rate. Ova seminated with rayed sperm showed varying
proportions of normally cleavage stages depending upon the
time of exposure of the sperm to the ultraviolet light. Long
exposures resulted in a preponderance of irregularly cleaved
ova. But even the regularly cleaved ova resulting from
seminations of sperm given short exposures were markedly re-
tarded when compared with the control ova in the other tube.
The ultraviolet treatment with the particular wavelength
used results presumably in the inactivation of the sperm
chromatin (see Swann and del Rosario, 1932), and depend-
ing on the time of exposure (e.g., intensity of radiation)
leaves the non-chromatic portions of the sperm relatively
unaffected. Dalq and Simon (1931) have shown that sperm
treated with ultraviolet light penetrate into the egg cyto-
plasm but pronucleus formation does not occur and the
chromatin disintegrates. If the sperm centrosome apparatus
is not inactivated normal cleavage occurs, otherwise irregular
development ensues.
The data of Pincus (1930) indicate that parthenogenetic
THE ACTIVATION OF UNFERTILIZED EGGS 111
cleavages occur later than normal cleavages (although the
time taken for the segmentation process itself is the same in
fertilized and unfertilized eggs) . It thus appears that the re-
tarded cleavages observed in vivo as the result of semination
with irradiated sperm are parthenogenetic in the sense that
the sperm chromatin did not participate in the mitoses.
Novak and Eisinger (1923) attempted to activate rabbit
eggs by tying off the tubes at the isthmus to prevent entry
of the ova into the uterus. The ova that they recovered
were either irregularly cleaved or fragmented with perhaps
one or two normal cleavages. Their data thus resemble those
of Mann (1924) on rat ova (see Table VIII) which do not
descend into the uterus in unmated animals. Grusdew
(1896) who injected sperm into the tops of rabbit tubes
together with ova from punctured follicles also tied the tubes
off at the isthmus and in a number of ova which gave no
evidence of sperm penetration he observed ordinarily ir-
regular but occasionally regular development. It would
seem then that parthenogenetic development may be in-
duced in vivo but that extensive embryonic differentiation
has not been demonstrated.
It is obvious, of course, that a mere beginning has been
made in the investigation of the parthenogenetic potencies of
tubal ova. Presumably normal embryos might develop if a
diploid cleavage nucleus could be induced to form. Pincus
and Enzmann (1935) have, in fact, found indications that
such a process may occur in activated rabbit eggs noting,
again, after a rather long latent period, two fusion nuclei in
unfertilized ova. The writer has observed an initial nuclear
division without cytoplasmic cleavage in a primate ovarian
o\nim cultured t/i vitro. For full development in vivo it
seems necessary that parthenogenetic ova should duplicate
with some exactitude not only the normal morphological
changes but also the rate of these processes. For the dif-
ferentiating embryo is dependent upon an uterine environ-
ment the optimum development of which involves a fairly
definite time schedule.
CHAPTER IX
THE GROWTH AND IMPLANTATION OF THE
BLASTODERMIC VESICLE
In the cinematographs of Lewis and Gregory (1929) the
regular cleavage of rabbit ova in vitro is shown to occur at
approximately the same rate as in vivo and the formation of
the blastocyst is initiated. The rapid expansion of the
blastocyst into the typical large blastodermic vesicle (see
Plate VII, Fig. 21) does not, however, occur. The attempted
expansion is apparently barred by the presence of the rela-
tively rigid zona pellucida and albumen coating so that the
blastocyst alternately expands and collapses over a period
of many hours until degeneration finally ensues. Br ache t
(1912, 1913) had previously shown that ova recovered from
the uterus of the rabbit at 5 to 6 days after coitus will
develop normally for 24 hours to 48 hours, passing from
the tridermic stage to the stage of the primitive streak,
with normal development of the ectoplacenta. Rabbit ova
enter the uterus between 72 and 75 hours after copulation
(Cruikshank, 1797; Assheton, 1894; Gregory, 1930) in the
early blastocyst stage and still surrounded by the zona
pellucida and the albumen coat. There is a rapid expansion
of the ovum at this time due to the infiltration of fluid into
the vesicle cavity so that by 96 hours after copulation the
blastocyst is easily three times the diameter of the tubal
egg. Very soon after the entry of the ovum into the uterus
the viscosity of the stretched albumen layer appears to
decrease so that its persistence about the large pre-primitive
streak vesicle of the 6th day must be due to a marked soften-
ing. By the end of the 6th day to the 7th day it disappears
completely due probably to its digestion by uterine fluids
since it does not disappear in culture-grown ova. The growth
112
VESICLE GROWTH AND IMPLANTATION 113
in culture of whole vesicles during the period when the
albumen and zona coverings still remain is extremely diffi-
cult for the ova soon degenerate and often collapse (Water-
man, 1932, 1934). As soon as the early primitive streak stage
is reached, explantation results in a moderate degree of de-
velopment. Waddington and Waterman (1933) explanted the
\
,!^ '
/ ■^-.
Fig. 30. Camera lucida drawings of embryonic
areas of the rabbit at the stages of explantation.
XG, late pre-primitive streak. XE, stage of pos-
terior thickening. XL, medium primitive streak.
XK, pre-somite. XB, three somite, p.st., prim-
itive streak; c.pl, chorda plate; p.kt., primitive
knot; p. pi., prochordal plate; p.m.s., pre-meso-
dermal somite; s., somite. (From the Journal of
Experimental Biology.)
embryonic portion of the blastodermic vesicles upon a me-
dium of chicken plasma plus chicken embryo extract and
found that the older and more differentiated the embryo at
the time of explantation the greater the degree of differenti-
ation in culture. Using the five stages illustrated in Figure 30,
the development observed was as follows :
(a) The stage of late pre-primitive streak gives no appar-
114 THE EGGS OF MAMMALS
ent differentiation as seen in whole mount preparations.
Localized thickenings only occur.
(b) The stage of posterior thickening and initial elongation
of the embryonic disc develops one or two beating hearts,
and localized thickenings after 4-5 days' growth in vitro.
(c) The stage of short primitive streak undergoes marked
elongation of the primitive streak and embryonic disc on the
2nd day; two, and in one case three, beating hearts appeared
after 2-3 days of culture.
(d) The stage of medium primitive streak gives results
comparable to (c) . In several instances brain, hearts, neural
tube and somites appear.
(e) The stage of long primitive streak gave rise to em-
bryos with as many as six pairs of somites after 1 day of
culture, and the pre-somite and two-somite stages give only
slightly, if at all, better development.
Nicholas and Rudnick (1934) similarly were unable to
obtain any adequate development of rat blastocysts in stages
earlier than the pre-somite or 5-7 somite. But vesicles in the
latter stages developed markedly in a medium consisting of
equal parts of rat plasma and 14-15 day rat embryo extract.
They report that growth occurs during the first twenty-four
hours in vitro gradually slowing and ceasing by the 36th hour.
''At 48 hours or earlier, differentiation in the embryo has
reached a maximum, at which it may be maintained for
another 24 hours.
''During this period the embryos in the best cases have
differentiated from 2 to 16 somites. The allantoic bud has
grown from a small lump of tissue at the angle between the
amnion and the posterior part of the embryo to join with the
superior surface of the ectoplacental cone. The heart, un-
formed at the time of implantation, has differentiated a two
chambered structure and has initiated its beat, the blood
islands have developed in the yolk sac epithelium, and cir-
culation has commenced, both in the yolk sac and in the
embryo. The nervous system has differentiated consider-
ably; eyes and ears have differentiated and the embryo as
VESICLE GROWTH AND IMPLANTATION 115
a whole has gone through a primary torsion, separating it
from the embryonic membranes in the region of the intestinal
portal and contributing to its apparent reversal of posture.
"The total growth attained in the 48 hour period is less
than half that attained by the normal embryo during the
same period. The maximum differentiation is nearly three-
quarters of that undergone by the normal. The factors
limiting growth are affected earlier than those limiting
differentiation.
'^ Apparently respiratory interchange is the most important
functional necessity at this stage. The efficiency of this
mechanism is not only lowered by the total absence of
maternal circulation but even further prevented by the
growth of a new enveloping membrane in the nature of a
decidua from the marginal cells of the ectoplacental cone.
The accumulation of break-down products due to metabolic
activity is another checking factor. A few preliminary
experiments have shown that these can be removed by
washing the entire culture in sterile Ringer's solution and
adding fresh embryonic extract. By using this method
embryos have been kept alive for 96 hours although growth
and differentiation occur only at a low rate during the last
24 hours."
Nicholas (1934) has also observed a few cases of the
development of rat embryos from ova dropped into the
uterine cavity, and extra-uterine pregnancies in man are of
course well known. In the rat the removal of the entire
gestation sac from the uterus into the peritoneal cavity
may be performed without hindering fairly advanced em-
bryo development in the extra-uterine environment (Selye,
Collip and Thomson, 19356).^ It therefore appears that
some somatic influence carries the ova through the critical
early blastocyst stages and that this influence does not
operate in the ordinary tissue culture media.
It will be recalled that this critical stage occurs at the
time of the disappearance of the egg envelopes and Hall
(1935) has recently presented data offering a possible clue
116 THE EGGS OF MAMMALS
to the critical events. He found that the zona pellucida of
rat and mouse ova placed in fluids of low acidity quickly
disappeared (at pH 3.7 or below). In a few cases the zona
pellucida was dissolved in Ringer's solution with a pH as
high as 5.4. Deciduomata of the rat have shown pH values
as low as 5.7, which are, however, not below the critical
levels of the in vitro experiments. Pincus and Enzmann
(unpublished data) have taken a number of measurements
of the pH of pseudopregnant and pregnant endometria and
Fig. 31. Left, normal rabbit blastocysts of the 5th day of
pregnancy. Right, blastocysts of the 5th day of pregnancy
from rabbit doe ovariectomized 18 hours after mating.
(From the American Journal of Physiology.)
have never observed pH values below 6.5. Nonetheless it
is possible that in the small decidual crypts into which the
ova fall the critical acidity may be attained.
Burdick and Pincus (1935) and Pincus and Kirsch (1936)
have examined this critical stage of development from a
somewhat different angle. Corner (1928) had noted that in
rabbit does in which both ovaries or all the corpora lutea
were removed shortly after fertilization the uterine ova
remained in the early blastocyst stage (see Figure 31 and
Tables XX to XXII), whereas in control rabbits with corpora
lutea normal development occurred. The degenerating
blastocysts were associated with an oestrus type of endo-
metrium, and normal growth of a progestational endome-
trium with implantation of embryos occurred when corpus
VESICLE GROWTH AND IMPLANTATION 117
luteum extracts were injected
daily after ovariectomy
(Allen and Corner, 1929).
Burdick and Pincus (1935)
observed that the daily in-
jection of oestrone begun
one or two days after copu-
lation in unoperated rabbits
(100-150 rat units per day)
and mice (5 rat units per
day) resulted in the degen-
eration of rabbit ova in the
early blastocyst stages and
of mouse ova in late morula
stages, i.e., at the stages
during which uterine entry
occurs. Pincus and Kirsch
(1936) extended these ob-
servations in order to fix the
critical time of action of the
hormone. Injections of oes-
trone were made at various
periods both before and after
ovulation, and in the case
of the post-0 vulatory injec-
tions the uteri were exam-
ined at the 10th to 12th
days to determine the extent
of implantation.
Their data presented in
Tables XXIII and XXIV
indicate clearly that the
minimum sterilizing dosage
can be given on days 3 to 4
post coitum. These days
cover the period of early
blastocyst development. The
TABLE XX
GROUP I BOTH OVARIES REMOVED AT 14-lH HR8. |
NO.
AUTOPSIED
STATE OF EMBRYOS
PROLIF.
1
4Hd
DEGENERATED 0.2 MM. DIAM.
18
4Kd
0.2 '<
34
4^d
0.15-0.2"
3
5Hd
0.2 "
2
7Hd
0.4 «<
4
7%d
0.3 ..
38
5%d
0,45 "
TABLE XXI
UKoUf U CONTROL OPERATIONS AT 1 j-18 V- HKS.
NO.
OP.
AUTOPSIED
STATE OF EMBRYOS
PROLIF.
33
oj-
ei^
7 NORMAL 0.5 MM.
+
24
01
eid
7 NORMAL O.C MM.
4 DEQEN.
■¥
27
m
5 rid
1 ABNORMAL 1 MM.
+
37
00
5Kd
7 NORMAL 2 MM.
+
23
08
G%d
3 NORMAL,
SHIELD STA3E
+
5
so
l%d
5 NORMAL,
iX SOMITES
+
21
SQ
md
1 NORMAL,
SOMITE 8TA0E
+
TABLE XXII
GROUP lU ALL CORPORA LUTEA EXCISED AT 15-20 HR3. |
NO.
OP.
AUTOPStED
STATE OF EMBRYOS
PROUF.
16
R. L.
m
4Hd
4 EARLY DEGEN.
0.4 MM.
\9
Qe
A%d
4UN8EQ.0VA
IN TUBE
30
fi§
h%d
NO EMB.
(OVULATION -(-)
'31
se
5Kd
..
32
so
h%d
..
10
08
V/2d
4 DEG. BLASTOCYSTS
0.2 MM. IN TUBE
-u
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118
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<M 00
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l-H
119
TABLE XXIV
The Effect of Various Types of Oestrone Injections during the
Preimplantation Period upon the Implantation Ratio. (From
Pincus and Kirsch, 1936)
Days
Rat
Total
Number
Number
Animal
AFTER
Units
Number
OF
OF Im-
Number
Mating
Injected
OF Rat
Corpora
planta-
Remarks
Injected
Daily
Units
Lutea
tions
16
1
200*
200
9
9
Implantations normal
17
1-2
200*
400
9
2
" "
18
1-3
200*
600
7
1
y) >>
20
1-3
200*
600
5
2
j> >>
19
1-4
200*
800
7
1
M >>
25
1-5
200 *
1000
10
24
4
200*
200
10
8
Implantations normal
38
4
400
400
7
1
)y >>
21
4-5
200
400
7
41
4-5
100
200
9
8
3 dying; 5 normal
26
4-6
200
600
8
44
5-6
200
400
12
7
Implantations subnormal
in size
48
5-6
200
400
To term
No litter
47
3-4
100
200
"
"
Litter of four
37
3-4
200
400
12
45
3-4
150
300
10
1 100%
71
3-4
150 t
300
11
dead
69
3-4
150 §
300
8
6
Implantations normal
40
3-4
100
200
14
5
Implantations
normal
60.3%
dead
52
3-4
100
200
13
6
2 embryos subnormal
60
3-4
75
150
8
1
Implantations
normal
87.5%
dead
56
3-4
373^
75
11
1
Implantations
normal
61
66
3-4
3-4
37H
371^
75
75
6
5
5
Implantations
normal
72.7%
dead
58
3-4
30
60
10
3
Implantations
normal
33.3%
65
3-4
30
60
11
11
Average diameter of
egg chambers 1.43
dead
59
3-4
25
50
9
7
X 1.07
Implantations
normal
62
3-4
25
50
10
10
Implantations
normal
11.1%
dead
64
3-4
25
50
8
7
Implantations
normal
13
1-5
3c.c.
.005%
15C.C.
6
5
Implantations
normal
54
No inj
NaOH
ections
10
10
1 subnormal in size
9.8%
dead
55a
"
"
5
5
Implantations normal
55b
n
n
11
8
,.
63
}f
>>
9
9
"
* Oestrone in aqueous solution (Parke-Davis Theelin).
t Crystalline oestrone in oily solution. § Crystalline oestrone in aqueous solution.
120
VESICLE GROWTH AND IMPLANTATION 121
minimum daily sterilizing dosage for days 3 and 4 is 150 rat
units of oestrone-in-oil. When lesser dosages are injected a
partially sterilizing effect is observed. This partially steriUz-
ing effect is measured by observing the ratio between the num-
ber of corpora lutea and the number of implantations. The re-
lation of the implantation ratio to the hormone dosage is given
in Figure 32. It will be seen that even relatively low hormone
dosages have a lethal effect upon a number of the embryos.
This effect may be due either to prevention of implantation
of vesicles developing normally till implantation time or to a
120 150
Fig. 32. Abscissa: oestrone dosage in R.U. per day. Ordinates: A, per cent
of embryos unimplanted; B, number of unimplanted embryos per female.
(From the American Journal of Physiology.)
degeneration before implantation. The latter alternative
seems most likely when one observes the degenerated condi-
tion of the preimplantation blastocysts. In addition prac-
tically all the embryos that do become implanted are normal
in appearance, and, in fact, give rise to normal young at
term (rabbit no. 47).
W^en eggs in the blastocyst stage are placed in culture
they will develop normally for 24 to 36 hours (Brachet, 1913;
Pincus, 1930). Cleaving ova will, as we have seen, develop
for several days and collapse when the blastocyst stage is
reached and presomite stages continue development for
3 to 9 days. This implies that the explanted blastocyst
either carries with it from the uterine environment a limited
supply of necessary nutrition or that it rapidly exhausts
the necessary materials from the ordinary culture medium.
122
THE EGGS OF MAMMALS
If oestrone in some way directly interferes with the assimila-
tion or metabolism of this critical nutrition then blastocysts
cultured with this hormone should show inhibited develop-
ment compared to that of controls in a normal medium.
Pincus and Kirsch (1936) cultured early blastocysts taken
from the uterus of rabbit does with varying amounts of
oestriol (12.5 to 25.2 y per culture) and found that control
blastocysts developed at the same time as those in the
oestriol-containing media. Oestriol was used instead of
oestrone because the former is much more soluble in aqueous
media and it also has a lethal effect upon developing blasto-
cysts when injected in vivo (see Table XXV). These experi-
ments show that the lethal effect of the hormone is not due
to the direct action of the hormone upon the developing
blastocyst. The sterilizing effects of oestriol and dihydro-
oestrone (Table XXV) indicate that the lethal effect is not
oestrone specific, and point again to the disturbance of a
needed nutritive condition.
TABLE XXV
The Effect of Various Injections of Oestriol and Dihydrooestrone
UPON the Implantation Ratio. (From Pincus and Kirsch, 1936)
Animal
Number
Days
AFTER
Mating
Injected
Amount
Injected
Daily
(IN
Gamma)
Total
Amount
(in
Gamma)
Number
OF
Corpora
Lutea
Number
of
Implan-
tations
Remarks
46
70
78
3-4
3-4
3-4
3-4
3-4
4-5
4-5
4-5
3-4
3-4
3-4
3-4
16.7*
16.7t
18.0t
22.2*
22.2t
11.1*
5.5*
11.1*
66.0§
100.0§
150.0§
225.0§
33.3
33.3
36.0
44.4
44.4
22.2
11.0
22.2
132.0
200.0
300.0
450.0
9
9
8
12
10
16
10
Tot
6
7
9
8
8
6
16
10
erm
6
3
2
Implantations normal
51
7."^
42
43
49
68
74
76
77
Implantations subnormal
in size
Implantations normal
No litter
Implantations normal
Average diameters of egg
chambers 1.90 X 1.43
cm.
Egg chambers = .8 X 1.0
and .9 X 1.1
* Dihydrooestrone in aqueous solution,
t Dihydrooestrone in oily solution.
§ Oestriol in oily solution.
VESICLE GROWTH AND IMPLANTATION 123
Just what special conditions are needed for carrying the
blastodermic vesicle over this critical stage cannot be ex-
plicitly stated. It is obvious that corpus luteum activity
is necessary for the establishment of these conditions, and
the oestrone effect is due to an inhibition of this activity.
Thus it is possible to overcome the partially sterilizing ef-
fect of low oestrone dosages by the simultaneous injection
of a corpus luteum hormone preparation (Pincus and Kirsch,
unpublished data). Other substances {e.g., vitamins A and
C) are ineffective as inhibitors of complete sterilization.
There seem to be tw^o alternatives: either (1) progesterone
or some corpus luteum product act directly upon the blasto-
cysts or (2) corpus luteum secretions induce a special uterine
environment through their action upon the endometrium.
Pincus and Enzmann (unpubhshed data) have made crude
extracts of the endometrium of pseudopregnant rabbit does,
and have cultured blastocysts in media containing these
extracts. No marked effect was obtained with the particular
preparations employed, but further investigation may dis-
close the presence of an active substance. It is certain that
blastocyst death due to oestrone action occurs in a uterus
the endometrium of which still shows at least partial pseudo^
pregnant proliferation. The minimum sterilizing dosage
employed by Pincus and Kirsch is insufficient to abolish
pseudopregnant growth completely (Leonard, Hisaw and
Fevold, 1931; Courrier and Raynaud, 1933). Courrier and
Raynaud (1934) have also found that dosages sufficient to
prevent implantation are below the level necessary for the
abolition of pseudopregnant growth. The data presented
here on sub-sterilizing dosages demonstrate explicitly that a
certain number of vesicles fail to develop in a uterus in
which others proceed normally. We may consider therefore
that there is necessary at least a threshold amount of a
necessary active substance, or an optimum-hydrogen ion
concentration alterations of which differentially affect the
various blastocysts, or a rate of uterine contraction which
causes the proper lodging of the blastocysts in the endo-
124 THE EGGS OF MAMMALS
metrium thus preventing their injury. The fact that blasto-
cysts in culture also show unusual sensitivity leaves the
first two of these alternatives.
The behavior and differentiation of the blastodermic ves-
icle at the time of implantation have been the object of
extensive investigation by mammalian embryologists since
the publication of Bischoff's (1852) classical memoir on the
subject. These investigations have been concerned chiefly
with presenting exact descriptions of the mode of implanta-
tion in various classes of mammals (see Robinson, 1904;
Grosser, 1909; Bonnet, 1903; Spee, 1915; Wilson, 1928;
Sansom and Hill, 1930) and the accompanying differentia-
tion of the vesicle. The physiological processes underlying
these phenomena have been scarcely investigated.
The writer has been interested in the phenomenon of
the delayed pregnancy which seems to offer an opportunity
to exploit the processes occurring at implantation. Delayed
pregnancy, or late parturition, occurs notably in the lactating
mouse or rat which is carrying a set of fertilized eggs during
lactation. This is a result of the fact that mice and rats
have an oestrus period within 48 hours of parturition in
which normal mating and fertilization take place. Enzmann,
Saphir and Pincus (1932) have analyzed all the available
data in the literature and found that in mice and rats each
suckling young on the average prolonged pregnancy by
about 21 hours (see Figure 33), though this time of prolonga-
tion seemed to vary somewhat from strain to strain. An
examination of mated mice in a series of timed matings
disclosed the fact that the preimplantation vesicle in suckling
females failed to implant at the normal time but some time
later depending upon the number of young suckling (see
Kirkham, 1916, 1918). Once implantation occurs the growth
of the embryo proceeds at the rate characteristic of normal
embryos (Enzmann, 1935). Obviously the lactation process
results in the establishment of conditions in uUro which
inhibit implantation, and the rather exact relationship be-
tween the degree of delay of pregnancy and the number of
VESICLE GROWTH AND IMPLANTATION 125
young suckling suggests that definite quantities of necessary
substances are withdrawn from the uterus as the result of
mammary gland activity.
Teel (1926) found that the daily injection of a NaOH
extract of the anterior hypophysis delayed implantation in
rats when injections were begun on the day of mating.
Injections on days 1 to 6 caused delayed implantation with
parturition occurring in normal fashion but several days
10
—
•4
9
—
^^
8
—
■
•
•
5'
—
■
•
!
t
W 4
—
14*
▲
SYMBOLS
3
•■ •■ 1
AVERAGES OF ALL DATA_ # |
A
GENE
TIMEI
RAL STOCK ▲
2
■> MATINGS ■
1
n
1
f (
1
1
1 1
I 1 1 1 I
4 5 6 7 8 9 10 11
NUMBER OF YOUNG SUCKLED
12 13 14
Fig. 33. Showing the relationship between the degree of delay of pregnancy
and the number of suckling young. (From the Anatomical Record.)
after term; injections on days 1 to 12 also caused delayed
implantation but a definite interference in the birth mecha-
nism so that only one of a series of females produced normal
hving young in a late parturition; injections over a longer
period resulted not only in delayed implantation but also
in stillbirths 5 to 7 days after normal term. The impairment
of the birth mechanism can therefore be avoided by early
injection and is presumably a phenomenon distinct from
that of delayed implantation. The inhibition of parturition
can be caused not only by alkaline pituitary extracts (Evans
and Simpson, 19296; Snyder, 1934) but also by corpus
126 THE EGGS OF MAMMALS
luteum extracts (Nelson, PMner and Haterius, 1930). Since
the pituitary extracts employed by Teel caused marked
luteinization of the ovaries of injected animals it is possible
that the delay in implantation may be due to excessive
corpus luteum secretion. Selye, CoUip and Thomson (1935??)
have ingeniously demonstrated that the rat ovary during
lactation presumably produces little or no oestrin, so that
the hormone-producing tissue of the ovary during lactation
is predominantly the luteal tissue. One need not postulate
hypersecretion by the corpus luteum during lactation but
merely an unbalance in which corpus luteum hormone pre-
dominates (thus Selye, Collip and Thomson actually obtain
larger corpora lutea in lactating mice when oestrin is
injected).
Wislocki and Goodman (1934) injected a preparation of
progestin (after Allen, 1930), for 8 days after mating into
two rabbits but no delay of pregnancy ensued. Antuitrin-S
and antuitrin-G injected in fairly large amounts during
early pregnancy were also ineffective although these prepara-
tions induced a fresh ovulation and new corpus luteum
formation. The ineffectiveness of progestin in the two experi-
ments of Wislocki and Goodman may have been due to an
insufficient dosage. On the other hand it is possible that
delayed pregnancy is due to an insufficiency of corpus luteum
secretion, so that the immediate effect of Teel's extract may
be considered the stimulation of oestrin production with
inhibition of luteal secretion followed by corpus luteum
activity which induced or completed the implantation proc-
ess. Hamlett (1935) is in fact of the opinion that delayed
implantation is due to hyposecretion of the corpus luteum.
He has found (1932) that copulation and cleavage occur
in the nine-banded armadillo of Texas during July, and the
unimplanted vesicle lies free in the uterine lumen until
early November when implantation takes place. Correlated
with the quiescent period is a large corpus luteum the cells
of which contain few or no secretory droplets or granules.
Shortly before implantation vacuolization and lipoidal secre-
VESICLE GROWTH AND IMPLANTATION 127
tion occurs in the luteal cell cytoplasm, and the removal
of such corpora lutea leads to abortion whereas removal
during the free vesicle period has no discernible effect upon
the uterus or ovum. Hamlett (1935) quotes a number of
instances of naturally-occurring delayed implantation of a
presumably similar nature.
This possibility has been tested by injecting oest rone-free
corpus luteum extracts into lactating pregnant mice during
the early part of pregnancy (unpublished data). Injections
of approximately l/20th of a Corner- Allen rabbit unit were
made over a 5 to 8 day period. A number of the mice failed
to produce any young but seven females gave birth to normal
litters. These were born not at term but much later; in
fact, the average date of birth was 4 days later than would
occur in delayed pregnancy if the expected delay is calculated
on the basis of 21 hours per suckling young.
The implication is clear that excessive corpus luteum
secretion caused a delay of pregnancy in mice. Since Teel
(1926) found that deciduomata formation could be readily
induced in the uteri of unmated females treated with his
extracts corpus luteum activity undoubtedly occurred as a
result of luteinizing hormone injection. The act of suckling
then, by prolonging corpus luteum activity (which it does —
see Parkes, 1929; Turner, 1932), results in a delay of im-
plantation. Selye and McKeown (1934a) have in fact shown
that suckling in rats prolongs pseudopregnancy and that
the effects of suckling do not occur in the absence of the
ovary (Selye and McKeown, 19346).
The fact that Teel obtained definite deciduomata in ani-
mals subjected to a treatment that produces delayed preg-
nancy indicates either: (1) that mechanical irritation is
more effective than ovum contact and that therefore the
corpus luteum effect is really subnormal or (2) that excessive
corpus luteum activity in some way inhibits the actual
process of implantation of the blastocysts. The problem is
an interesting one and is receiving further investigation.
CHAPTER X
SUMMARY AND RECAPITULATION
For the purposes of this monograph an ovum is considered
as such from the moment of its functional differentiation in
the ovary until its implantation in the uterine endometrium.
An examination has been made of the experimental investi-
gations of the growth and development of the mammahan
ovum during the various stages of its life history in the
ovary and oviducts.
The problem of the origin of the definitive ova has re-
ceived much attention, but it cannot be said to have been
completely resolved. If we are to judge by evidence from
non-mammalian forms the large amoeboid primordial germ
cells must enter the embryonic gonad if it is to differentiate
as a functional organ. A functional ovary develops only
from embryonic gonads in which the secondary sex cords
proliferate to form a true ovarian cortex associated with the
germinal epithelium.
The ovaries of young mammals contain large numbers of
primitive oocytes. The conception that these oocytes are
the only precursors of the definitive ova is controverted by
a large body of recent evidence which indicates that new
ova are .proliferated from the germinal epithelium and that
the rate of this proliferation varies with the various stages
of the oestrus and pregnancy cycles. Ovogenesis in the adult
seems to be partially inhibited by certain secretions of the
anterior pituitary, the gonad-stimulating hormones affect-
ing follicle growth primarily. The exact relation of the
gonad-stimulating hormones to the ovogenetic processes is
not at all obvious. It seems certain that the prophase
stages of the oocyte nuclei occur independent of pituitary
hormone activity.
128
SUMMARY AND RECAPITULATION 129
Pituitary hormones are definitely concerned in the final
stage of ovum maturation, the first polar division which
normally occurs in the ovary of most mammals. The pi-
tuitary secretions do not affect the eggs directly but initiate
changes in the follicles which make for maturation in the ova.
Similar changes occur in atretic follicles with a resulting
"pseudomaturation" in the ova of such follicles. The initia-
tion of ovum activation represented by the first maturation
division occurs in vitro simply upon the explantation of
ovarian eggs. Maturation in vivo and in vitro can be ex-
plained as the result of a functional isolation of the ovum
from the follicular epithelium. It is held probable therefore
that the parthenogenetic development of ova observed in
mammalian ovaries occurs as the result of the establishment
in the follicle of special activating conditions.
Parthenogenetic development of unfertilized tubal ova
rarely if ever occurs in vivo. In most eutherian mammals the
eggs are shed surrounded by follicle cells. If sperm are not
present the surrounding cells slowly fall away, and the
naked ova descend into the lower portion of the tubes
where they degenerate and are eventually either resorbed
or washed out into the uterus. When sperm are present
there is a rapid dissolution of the surrounding follicle cells
due to the action of a heat labile substance carried by the
sperm. It has been claimed that this same substance acti-
vates the ova into forming the second polar body, but the
available evidence is contradictory. Tubal eggs remain
fertilizable for a few hours in the rabbit, and for thirty
hours in the ferret.
Manamalian ova may be fertilized in vitro and normal
cleavage ensues. This is most readily demonstrated with
rabbit ova, for the ova of most of the other forms examined
do not cleave or develop appreciably under the ordinary
conditions of tissue culture. Segmentation in vivo occurs at
fairly characteristic rates in the various species of mammals.
The cleavage rate in rabbits is definitely correlated with the
adult size of the strain employed. The cleavage process
130 THE EGGS OF MAMMALS
itself is under the control of a cyanide-labile system. The
process of cleavage is apparently independent of the activity
of the primary sex hormones, oestrin and progestin.
Tubal rabbit ova readily exhibit parthenogenetic cleav-
ages under certain conditions of explantation in vitro. Parthe-
nogenetic activation can be initiated experimentally by treat-
ment with cytolytic agents, by exposure to hypertonic
solutions, and by heat treatment.
The development of the blastodermic vesicle in vivo is
conditioned by the activity of corpus luteum secretions. In
the absence of the corpus luteum development does not
occur beyond that stage in which ova just entering the uterus
are found. The evidence indicates that the corpus luteum
secretions either stimulate the eggs directly or provide
through stimulation of the uterine endometrium a suitable
environment for the developing blastocysts. Oestrin and
allied compounds prevent blastocyst growth by inhibiting
the corpus luteum effect, the ova being most sensitive to
this inhibition during the early blastocyst stages.
The implantation process itself is also under hormonal
control. In the rat and mouse ovum implantation is delayed
during lactation. This delay appears to be due to excessive
corpus luteum secretion.
The development of various techniques for the explanta-
tion of ova both in vivo and in vitro makes available a variety
of experimental investigations of the manmaalian ovum.
The ova of certain forms are particularly adapted to experi-
mental manipulation. Mammalian ova normally develop in
a homeostatic environment. Certain components of this
homeostasis sharply limit the extent and nature of ovum
development at certain stages. During other phases of its
growth the ovum appears to be a relatively independent
organism. Careful investigation of the physiological proc-
esses occurring in the ovum itself and in its homeostatic
environment is made possible by the various explantation
and transplantation techniques.
BIBLIOGRAPHY
Addison, W. H. F. 1917. Fragmentation of the ovum within the Graafian
follicle. Proc. Path. Soc. Phila. 37, 1.
Allen, B. M. 1904. Embryonic development of the ovary and testis of
the mammals. Am. J. Anat. 3, 89.
Allen, E. 1923. Ovogenesis during sexual maturity. Am. J. Anat. 31,4:39.
. 1932. The ovarian follicular hormone, theelin; animal reactions.
In: Sex and internal secretions. Williams and Wilkins. Baltimore.
Allen, E., Francis, B. F., Robertson, L. L., Colgate, C. E., Johnston,
C. G., DoisY, E. A., KouNTz, W. B. and Gibson, H. V. 1924. The
hormone of the ovarian follicle; its localization and action in test
animals and additional points bearing upon the internal secretion of
the ovary. Am. J. Anat. 34, 133.
Allen, E., Kountz, W. B. and Francis, B. F. 1925. Selective elimination
of ova in the adult ovary. Am. J. Anat. 34, 445.
Allen, E., Pratt, J. P., Newell, Q. U. and Bland, L. J. 1930a. Human
tubal ova; related early corpora lutea and uterine tubes. Carnegie
Inst. Wash. Contrib. Emhryol. 22, 45.
. 19306. Human ova from large follicles; including a search for mat-.
uration divisions and observations on atresia. Am. J. Anat. 46, 1.
Allen, W. M. 1930. Physiology of the corpus luteum; preparation and
some chemical properties of progestin, hormone of corpus luteum
which produces progestational proliferation. Am. J. Physiol. 92, 174.
Allen, W. M. and Corner, G. W. 1929. Physiology of the corpus luteum
III. Normal growth and implantation of embryos after very early
ablation of the ovaries, under the influence of extracts of the corpus
luteum. Am. J. Physiol. 88, 340.
Amann, J. A. 1899. Ueber Bildung von Ureiern und primarfollikelahn-
lichen Gebilden in senilen Ovarium. Festschr. zum siebzigsten Ge-
burtstag C. Kupfer.
Anderson, D. H. 1927. The rate of passage of the mammalian ovum
through various portions of the Fallopian tube. Am. J. Physiol. 82,
557.
Arai, H. 1920a. Post natal development of the ovary in the white rat.
Am. J. Anat. 27, 405.
. 19206. On the cause of the hypertrophy of the surviving ovary
131
132 THE EGGS OF MAMMALS
after semi-spaying and the number of ova in it. Am. J. Anat.
28, 59.
AsAMi, G. 1920. Observations on follicular atresia in the rabbit ovary.
Anat. Rec. 18, 323.
Aschner, B. 1914. Ueber den Kampf der Teile im Ovarium. Arch. Ent.
Mech. 40, 565.
AsDELL, S. A. 1924. Some effects of unilateral ovariectomy in rabbits.
Brit. J. Exp. Biol. 1, 473.
AssHETON, R. 1894. A re-investigation into the early stages of the de-
velopment of the rabbit. Quar. J. Micr. Sc. 37, 113.
Athias, M. 1909. Les phenomenes de division de I'ovule dans les follicules
de De Graaf en voie d'atresie chez le Lerot. Anat. Anz. 34, 1.
. 1920. Invagination de I'epithelium superficiel et neoformation
ovulaire dans I'ovaire transplante chez le cobaye. Compt. rend. Soc.
biol. 83, 1647.
VON Baer, K. E. 1827. De ovi mammalium et hominis genesi. Epistola.
Leopoldi Vossi. Leipzig.
Balfour, F. M. 1878. On the structure and development of the verte-
brate ovary. Quar. J. Micr. Sc. 18, 383.
. 1882. Handbuch der vergleichenden Embryologie. Trans, of
Vetter. Jena.
Barry, M. 1838. Researches in embryology, first series. Phil. Trans. Roy.
Soc. London, p. 301.
. 1839. Researches in embryology, second series. Phil. Trans. Roy.
Soc. London, p. 307.
Bellerby, C. W. 1929. The relation of the anterior lobe of the pituitary
to ovulation. /. Physiol. 67, xxxiii.
Benoit, J. 1930. Contribution a I'etude de la lignee germinale chez le
poulet. Destruction precoce des gonocytes primaires par les rayons
ultraviolets. Compt. rend. Soc. biol. 104, 1329.
BiEDL, A., Peters, H. and Hofstatler, R. 1922. Transplantation
befruchteter Eier bei Kaninchen. Z. Geburtsh. 84, 60.
BiscHOFF, T. L. W. 1842. Entwicklungsgeschichte der Saugethiere und
des Menschen. Voss. Leipzig.
. 1845. Entwicklungsgeschichte des Hundeeies. Freidrich Vie wag
und Sohn. Braunschweig.
. 1852. Entwicklungsgeschichte des Meerschweinchens. J. Rickersche
Buchhandlung. Giessen.
. 1854. Entwicklungsgeschichte des Rehes. J. Rickersche Buch-
handlung. Giessen.
BIBLIOGRAPHY 133
Bond, C. S. 1906. Some points in uterine and ovarian physiology and
pathology in rabbits. Brit. Med. J. 2, 121.
Bonnet, R. 1884. Beitrage zur Embryologie der Wiederkauer, gewonnen
am Schafei. Arch. Anal. u. Physiol. 170.
. 1891. Grundriss der Entwickelungsgeschichte der Haussaugethiere.
Verlag von Paul Parey. Berlin.
. 1899. Gibt es bei Wirbeltieren Parthenogenesis? Ergeb. Anat.
Entwcklngsgesch. 9, 820.
. 1903. Uber Syncytien, Plasmodien und Symplasma in der Placenta
der Saugetiere und des Menschen. Monats. Geburtsh. u. Gynak. 18, I.
BosAEUs, W. 1926. Beitrage zur Kenntnis der Genese der Ovarialem-
bryoma. Almquist und Wiksells. Upsala.
Bracket, A. 1912. Developpement in vitro de blastodermes et de jeunes
embryons de mammiferes. Compt. rend. Soc. biol. 155, 1191.
. 1913. Recherches sur le determinisme hereditaire de Toeuf des
Mammiferes. Developpement "in vitro" de jeunes vesicules blasto-
dermiques du lapin. Arch. Biol. 28, 447.
Brambell, F. W. R. 1927. The development and morphology of the
gonads of the mouse. Part I. The morphogenesis of the indifferent
gonad and of the ovary. Proc. Roy. Soc. B 101, 391.
. 1927. The development and morphology of the gonads of the
mouse. Part II. The development of the Wolffian body and ducts.
Proc. Roy. Soc. B 102, 206.
. 1928. The development and morphology of the gonads of the
mouse. Part III. The growth of the follicles. Proc. Roy. Soc. B 103,
258.
. 1930. The development of sex in vertebrates. Macmillan Co.
New York.
Brambell, F. W. R., Parkes, A. S. and Fielding, V. 1927a. Changes
in the ovary of the mouse following exposures to x-rays. I. Irradiation
at three weeks old. Proc. Roy. Soc. B 101, 29.
. 1927b. Changes in the ovary of the mouse following exposures to
x-rays. II. Irradiation at or before birth. Proc. Roy. Soc. B 101, 95.
Branca, A. 1925. L'ovocyte atresique et son involution. Arch. Biol. 35,
325.
Buhler, a. 1894. Beitrage zur Kentniss der Eibildung beim Kaninchen
und der Markstrange des Eierstockes beim Fuchs und Menschen.
Z. wissensch. Zool. 58, 314.
BuRDicK, H. 0. and Pincus, G. 1935. The effect of oestrin injections
upon the developing ova of mice and rabbits. Am. J. Physiol. 111,201.
134 THE EGGS OF MAMMALS
Butcher, Earl 0. 1927. The origin of the definitive ova in the white
rat (Mus norvegicus albinus). Anat. Rec. 37, 13.
. 1932. Regeneration in hgated ovaries and transpla^.ted ovarian
fragments of the white rat (Mus norvegicus albinus). Anat. Rec. 54,
87.
BuYSE, Adrian. 1935. The differentiation of transplanted mammalian
gonad primordia. J. Exp. Zool. 70, 1.
Caldwell, W. H. 1887. The embryology of the Monotremata and Mar-
supalia. Part I. Phil. Trans. Roy. Soc. B 178, 463.
Campbell, J. A. 1924. Changes in the tensions of CO2 and O2 in gases
injected under the skin and into the abdominal cavity. J. Physiol. 59,
1.
Carmichael, E. S. and Marshall, F. H. A. 1908. On the occurrence of
compensatory hypertrophy in the ovary. J. Physiol. 36, 431.
Carrel, A. 1924. Leucocytic trephones. /. Am. Med. Assoc. 82, 255.
Casida, L. E. 1935. Prepuberal development of the pig ovary and its re-
lation to stimulation with gonadotropic hormones. Anat. Rec. 61,
389.
Castle, W. E. and Gregory, P. W. 1929. The embryological basis of
size inheritance in the rabbit. J. Morph. and Physiol. 48, 81.
Champy, C. 1927. Parthenogenese experimentale chez le Lapin. Compt.
rend. Soc. hiol. 96, 1108.
Chapin, Catherine L. 1917. A microscopic study of the reproductive
system of foetal free-martins. /. Exp. Zool. 23, 453.
Charlton, H. H. 1917. The fate of the unfertilized egg in the white
mouse. Biol. Bull. 33, 321.
Clark, E. B. 1923. Observations on the ova and ovaries of the guinea-
pig, Cavia cobaya. Anat. Rec. 25, 313.
Clark, R. T. 1934. Studies on the physiology of reproduction in the
sheep. II. The cleavage stages of the ovum. Anat, Rec. 60, 135.
Coert, H. J. 1898. Over de ontwikeling en den bouw van den geslacht-
sklier bijde zoogdiern meer in het bijzonder van den eierstock. Akad.
Proefschrift. Leiden.
Corey, E. L. 1928. Effect of prenatal and postnatal injections of the
pituitary gland in the white rat. Proc. Soc. Exp. Biol. & Med. 25, 498.
Corner, G. W. 1928. Physiology of the corpus luteum. I. The effect of
very early ablation of the corpus luteum upon embryos and uterus.
Am. J. Physiol. 86, 74.
Courrier, R. 1923. Vesicule blastodermique parthenogenetique dans un
ovaire de cobaye impubere. Arch. Anat., Histol. et Embryol. 2, 455.
BIBLIOGRAPHY 135
CouRRiER, R. et Oberling, C. 1923. Parthenogenese spontanee dans
I'ovaire du cobaye. Bull. Soc. Anat. Paris 93, 724.
CouRRiER, R. et Raynaud, R. 1933. Experiences d'antagonisme hu-
moral ovarien realisees avec I'etalon international de folliculine cris-
tallisee. Compt. rend. Soc. biol. 115, 299.
. 1934. Etude quantitative de Tavortement folliculinique provoque
chez la lapine, par Thormone cristallisee. Realisation d'un avortement
partiel. Compt. rend. Soc. biol. 116, 1078.
CowPERTHWAiTE, Marian M. 1925. Observations on pre- and post-
pubertal oogenesis in the white rat (Mus norvegicus albinus). Am. J.
Anat. 36, 69.
Crew, F. A. E. 1927. On the effects of unilateral ovariotomy and sal-
pingectomy in the rat. Biol. Generalis 3, 207.
Cruikshank, W. 1797. Experiments in which, on the third day after
impregnation the ova of rabbits were found in the fallopian tubes
and on the fourth day after impregnation in the uterus itself; with
the first appearance of the foetus. Phil. Trans. Roy. Soc. London 18,
197.
Dalq, a. et Simon, S. 1931. Contribution a I'analyse des fonctions
nucleaires dans I'ontogenese de la Grenouille. III. Etude statistique
et cytologique des effets de I'irradiation d'un des gametes sur la
gastrulation chez Rana Fusca. Arch. Biol. 1^2, 107.
Davenport, C. B. 1925. Regeneration of ovaries in mice. J . Exp. Zool.
Deansley, R., Fee, A. R. and Parkes, A. S. 1930. Studies on ovulation.
II. The effect of hypophysectomy on the formation of the corpus
luteum. /. Physiol. 70, 38.
Defrise, a. 1933. Some observations on living eggs and blastulae of the
albino rat. Anat. Rec. 57, 239.
DoiSY, E. A., Curtis, J. and Collier, W. D. 1931. Effect of theelin
upon the developing ovary of the rat. Proc. Soc. Exp. Biol. & Med. 28,
885.
DoMM, L. V. 1929. Spermatogenesis' following early ovariotomy in the
brown leghorn fowl. Arch. Eritwcklngsmech. Org. 119, 171.
DoRAN, M. A. 1902. Pregnancy after removal of both ovaries for cystic
tumor. /. Ohst. a7id Gynec. Brit. Emp. 2, 1.
Emery, F. E. 1931. Changes in the ovary and oestrus cycle following
the removal of one ovary in albino rats. Physiol. Zool. 4, 101-
Engle, E. T. 1927a. Polyovular follicles and polynuclear ova in the
mouse. Anat. Rec. 35, 341.
136 THE EGGS OF MAMMALS
Engle, E. T. 19276. A quantitative study of follicular atresia in the
mouse. Am. J. Anat. 39, 187.
. 1928. The role of the anterior pituitary in compensatory ovarian
hypertrophy. Anat. Rec. 37, 275.
. 1929. Ovarian responses: differences elicited by treatment with
urine from pregnant women and by freshly implanted anterior lobe.
J. Am. Med. Assoc. 93, 276.
. 1931a. Prepubertal growth of the ovarian follicle in the albino
mouse. Anat. Rec. 4-8, 341.
. 19316. The pituitary-gonadal relationship and the problem of pre-
cocious sexual maturity. Endocrinology 15, 405.
Enzmann, E. V. 1935. Intrauterine growth of albino mice in normal and
in delayed pregnancy. Anat. Rec. 62, 31.
Enzmann, E. V., Saphir, N. R. and Pincus, G. 1932. Delayed preg-
nancy in mice. Anat. Rec. 5^, 325.
Evans, H. M. and Cole, H. H. 1931. An introduction to the study of
the oestrus cycle in the dog. Mem. Univ. Calif. 9, 65.
Evans, H. M., Meyer, K. and Simpson, M. E. 1932. Relation of prolan
to the anterior hypophyseal hormones. Am. J. Physiol. 100, 141.
Evans, H. M., Meyer, K., Simpson, M. E., Szarka, A., Pencharz, R. I.,
Cornish, R. W. and Reichert, F. L. 1933. The growth and gonad-
stimulating hormones of the anterior hypophysis. Mem. Univ. Calif.
11,1.
Evans, H. M. and Simpson, M. E. 1928. Antagonism of growth and sex
hormones of the anterior hypophysis. /. Am. Med. Assoc. 91, 1337.
. 1929a. A comparison of the ovarian changes produced in immature
animals by implants of hypophyseal tissue and hormone from the
urine of pregnant women. Am. J. Physiol. 89, 381.
' . 19296. Impairment of the birth mechanism due to hormones from
the anterior hypophysis. Proc. Soc. Exp. Biol. & Med. 26, 595.
Evans, H. M. and Swezy, 0. 1931. Ovogenesis and the normal follicular
cycle in adult mammalia. Mem. Univ. Calif. 9, 119.
Feltx, W. 1912. Development of the urogenital organs, in: Manual of
human embryology, 2, 752. Keibel and Mall. Philadelphia.
Fellner, 0. 0. 1909. Histologie des Ovariums in der Schwangerschaft.
Arch. mikr. Anat. 73, 288.
Fevold, H. L. and Hisaw, F. L. 1934. Interactions of gonad stimulating
hormones in ovarian development. Am. J. Physiol. 109, 655.
Fevold, H. L., Hisaw, F. L. and Greep, O. 1934. Factors which govern
ovarian development. Anat. Rec. 60 Supp., 51.
BIBLIOGRAPHY 137
Fevold, H. L., Hisaw, F. L. and Leonard, S. L. 193 L The gonad-
stimulating and the luteinizing hormones of the anterior lobe of the
hypophysis. A??i. J. Physiol 97, 291.
FiRKET, J. 1920. On the origin of germ cells in higher vertebrates. Anat.
Rec. IS, 309.
Fischer, Albert. 1925. Tissue culture. Levin and Munksgaard. Copen-
hagen.
Flemming, W. 1885. Tiber die Bildung von Richtungsfiguren in Sauge-
tiereiern beim Untergang Graafscher Follikel. Arch. Anat. u. Eni-
wickl. 221.
FouLis, J. 1876. The ova and ovary in man and other mammalia. Quar.
J. Micr. Sc. 16, 190.
Friedgood, H. and Pincus, G. 1935. Studies on the conditions of activ-
ity in endocrine organs. XXX. The nervous control of the anterior
pituitary as indicated by maturation of ova and ovulation after
stimulation of cervical sympathetics. Endocrinology 19, 710.
Friedman, M. H. 1929. Mechanism of ovulation in the rabbit. II. Ovu-
lation produced by the injection of urine from pregnant women.
Am. J. Phijsiol. 90, 617.
Fuss, A. 1911. Uber extraregioniaire Geschlechtzellen bei einem Men-
schhchen Embryo von vier wochen. Anat. Am. 39, 407.
. 1913. Uber die Geschlechtzellen des Menschen und der Saugetiere.
Arch. mikr. Anat. 81, 1.
Genther, I. 1931. Irradiation of the ovaries of guinea pigs and its effect
on the oestrus cycle. Am. J. Anat. 48, 99.
. 1934. X-irradiation of the ovaries of guinea pigs and its effect on
subsequent pregnancies. Am. J. Anat. 55, 1.
Gerard, P. 1920. Contribution a I'etude de Fovaire des mammiferes.
L'ovaire de Galago mossambicus (Young). Arch. Biol. 30, 357.
Gilchrist, F. and Pincus, G. 1932. Living rat eggs. Anat. Rec. 54,
275.
Gordon, C. S. 1896. Two pregnancies following removal of both ovaries
and ligation of tubes. Trans. Am. Gynec. Soc. 21, 104.
Gregory, P. W. 1930. The early embryology of the rabbit. Carnegie
Inst. Wash. Contrib. to Embryol. 21, 141.
Gregory, P. W. and Castle, W. E. 1931. Further studies on the em-
bryological basis of size inheritance in the rabbit. /. Exp. Zool. 59,
199.
Grosser, 0. 1909. Vergl. Anat. u. Entwicklung der Eihaute und der
Placenta. Wilh. Braumiiller. Wien u. Leipzig.
138 THE EGGS OF MAMMALS
Grusdew, W. S. 1896. Versuche liber die kiinstliche Befruchtung von
Kanincheneiern. Arch. Anat. u. Physiol., Anat. Abt. 269.
GuRWiTSCH, A. 1900. Idiozom und Centralkorper im Ovarialei der
Saugetiere. Arch. mikr. Anat. 66, 377.
GuTHERZ, S. 1925". Tiber vorzeitige Chromatinreifung an physiologisch
degenerierenden Saugeoozyten des friihen Wachstumsperiode. Z.
mikr. Anat. Forsch. 2, 1.
Haberlandt, G. 1922. Uber Zellteilungshormone und ihre Beziehungen
zur Wimdheilung, Befruchtung, Parthenogenesis und Adventivem-
bryone. Biol. Zentr. Jf2, 145.
Haggstrom, p. 1922. Uber degenerative parthenogenetische Teilungen
von Eizellen in normalen Ovarien des Menschen. Acta Gynecol.
Scandinav. 1, 137.
Hall, B. V. 1935. The reactions of rat and mouse eggs to hydrogen
ions. Proc. Soc. Exp. Biol. & Med. 32, 747.
Hamlett, G. W. D. 1932. The reproductive cycle in the armadillo.
Z. wissensch. Zool. 1^1, 143.
. 1935. Delayed implantation and discontinuous development in
the mammals. Quar. Rev. Biol. 10, 432.
Hammond, J. 1928. Die Kontrolle der Fruchtbarkeit bei Tieren. Zilch-
tungskunde 3, 523.
. 1934. The fertilization of rabbit ova in relation to time. A method
of controlling the litter size, the duration of pregnancy, and the weight
of the young at birth. J. Exp. Biol. 11, 140.
Hammond, J. and Asdell, S. A. 1926. The vitahty of spermatozoa in
the male and female reproductive tract. Brit. J. Exp. Biol. 4, 155.
Hammond, J. and Marshall, F. H. A. 1925. Reproduction in the rabbit.
Oliver and Boyd. London.
Hammond, J. and Walton, A. 1934. Notes on ovulation and fertilization
in the ferret. /. Exp. Biol. 11, 307.
Hanson, F. B. and Boone, C. 1926. On the migration of ova from one
uterine horn to the other in the albino rat. Am. Nat. 60, 257.
Hargitt, G. T. 1925. The formation of the sex glands and germ cells
of mammals. 1. The origin of the germ cells in the albino rat. /.
Morphol. and Physiol. Jfi, 517.
. 1930. The formation of the sex glands and germ cells of mammals.
V. Germ cells in the ovaries of adult pregnant and senile albino
rats. /. Morphol. and Physiol. 50, 453.
Harms, J. W. 1926. Korper und Keimzellen. J. Springer. Berlin.
Hartman, C. G. 1916. Studies in the development of the opossum,
BIBLIOGRAPHY 139
Didelphys virginiana L. I. History of early cleavage. 11. Formation
of the blastocyst. /. Morphol. 27, 1.
Hartman, C. G. 1919. Studies in the development of the opossum,
Didelphys virginiana L. HI. Description of new material on matura-
tion, cleavage, and endoderm formation. IV. The bilaminar blas-
tocyst. /. Morphol. 32, 1.
. 1924. Observations on the viability of the mammalian ovum.
A7n. J. Obst. and Gij.iec. 7, 1.
. 1925. Observations on the functional compensatory hypertrophy
of the opossum ovary. Am. J. Anat. 35, 1.
. 1929. How large is the mammalian egg? A review. Quar. Rev.
Biol. 4, 373.
. 1932a. Studies in the reproduction of the monkey, Macacus (Pithe-
cus) rhesus, with special reference to menstruation and pregnancy.
Carnegie Inst. Wash. Contrib. Embryol. 23, 1.
. 19326. Ovulation and the transport and viabihty of ova and sperm
in the female genital tract. In Allen: Sex and internal secretions.
Williams and Wilkins. Baltimore.
Harz, W. 1883. Beitrage zur Histologie des Ovariums der Siiugetiere.
Arch. mikr. Anat. 22, 374.
Hatai, S. 1913. The effect of castration, spaying, or semi-spaying on the
weight of the central nervous system and of the hypophysis of the
albino rat; also the effect of semi-spaying on the remaining ovary.
/. Exp. Zool. 15, 297.
. 1915. The growth of organs in the albino rat as affected by gonad-
ectomy. /. Exp. Zool. 18,1.
Haterius, H. 0. 1928. An experimental study of ovarian regeneration
in mice. Physiol. Zool. 1, 45.
Heape, W. 1883. The development of the mole, Talpa Europea. The
formation of the germ layers and early development of the medullary
groove and notochord. Qttar. J. Micr. Sc. 23, 412.
. 1886. The development of the mole (Talpa Europea) ; the ovarian
ovum and segmentation of the ovum. Quar. J. Micr. Sc. 26, 157.
. 1905. Ovulation and degeneration of ova in the rabbit. Proc. Roy.
Soc. B 76, 260.
Hegner, R. W. 1914. The germ-cell cycle in animals. Macmillan Co.
New York.
Henneguy, F. 1893. Sur la fragmentation parthenogenesique des ovules
des vertebres pendant I'atresie des follicules de Graaf. Compt. rend.
Soc. biol. 45, 500.
140 THK rXlGS OF MAMMALS
Hensen, V. 1869. Uber die Ziiclitunfi; unhernichteter Eier. Centr. med.
Wissemch. 7, 403.
. 1870. Beobachtung fiber die Befruchtung iind Entvvicklung des
Kaninchens und Meerschweinchens. Z. Anal. Eniwckimjs. 1, 213; 851.
Hertz, R. and IIisaw, F. L. 1934. l*]ffects of follicle-stinuilating and
luteinizing pituitary extracts on the ovaries of the infantile and
juvenile rabbit, jlm. J. Physiol. lOS, 1.
Heuser, C. H. and Streeter, G. L. 1929. Early stages in the develop-
ment of pig embryos. Carnegie Inst. Wash. Conirih. Embryol. 20, 1.
IIeys, Florence. 1929. Does regeneration follow complete ovariotomy
in the albino rat? Science 70, 289.
. 1931. The problem of the origin of the germ cells. Quar. Rev.
Biol. 6, 1.
Hill, J. P. 1910. The early development of the Marsupalia with especial
reference to the native cat (Dasyurus viverrinus). Quar. J. Micr.
Sc. 56, 1.
. 1918. Some observations on the early development of Didelphys
aurita. Quar. J. Micr. Sc. OS, 91.
Hill, J. P. and Tuihe, M. 1924. The early development of the cat
(Felis domestica). Quar. J. Micr. Sc. 6S, 513.
Hill, M. and Parkes, A. S. 1931. Attempts to promote the reformation
of germ cells. /. Anat. 65, 212.
HiNDLE, E. 1910. A cytological study of artificial parthenogenesis in
Strongylocentrotus purpuratus. Arch. Entwcklngsmechn, 31, 145.
HiNSEY, J. C. and Markee, J. Vj. 1933. Studios on prolan-induced ovula-
tion in midbrain and midbrain-hypophysectomized rabbits. Am. J.
Physiol. 106, 48.
HiSAW, F. L., Fevold, H. L., Foster, M. A. and Hellbaum, A. A. 1934.
A physiological explanation of the oestrus cycle of the rat. Anat,
Rec. 60 Sup p., 52.
HuBER, G. C. 1915. The development of the albino rat, Mus norvegicus
albinus. I. From the pronuclear stage to the stage of the mesoderm
anlage; end of the first to end of the ninth day. /. Morphol. 26, 247.
HuBRECHT, A. A. W. 1912. Friihe Entwickhmgsstadien des Igels und ihre
Bedeutung fiir die Vorgeschichte (Phylogenese) des Amnions. Zool.
Jahrb. Supp. XV, 2, 739.
Humphrey, R. R. 1928. The developmental potencies of the intermediate
mesoderm of amblystoma when transplanted into ventro-lateral sites
in other embryos: the primordial germ cells of such grafts and their
role in the development of a gonad. Anat. Rec. Jfi, 67.
BIBLIOGRAPHY 141
Janosik, J. 1897. Die Atrophic der FoIIikel und ein seltsames Verhalten
der Eizelle. Arch. mikr. Anal. 48, 169.
Jenkinson, J. W. 1900. A reinvestigation of the early stages of the de-
velopment of the mouse. Quar. J. Micr. Sc. 43, Gl.
. 1913. Vertebrate embryology. Oxford.
Jones, T. W. 1837. On the first changes in the ova of Mammifera in
consequence of impregnation, and the mode of origin of the chorion.
Phil. Trans. Roy. Sac. 2, 339.
. 1838. On the ova of man and mammiferous brutes as they exist
in the ovaries l^efore impregnation and on the discovery in them of a
vesicle. London Med. Gaz., 680.
. 1885. On the ova of man and mammals before and after fecunda-
tion. Lancet 2, 283.
Just, E. E. 1928. Cortical reactions and attendant physico-chemical
changes in ova following fertilization. In Alexander: Colloid Chem-
istry. The Chemical Catalog Co. Inc. New York.
Kampmeier, O. F. 1929. On the problem of "parthenogenesis" in the
mammalian ovary. Am. J. Anal. 43, 45.
Kanel, V. Y. 1901. Regeneration processes in the ovaries of rabbits.
Mattissen. Kieff.
Keibel, F. 1888. Zur Entwicklungsgeschichte des Igels. Anat. Am. 3,
632.
. 1894. Studien zur Entwicklungsgeschichte des Schweines (Sus
scrofa dom.). Schwalbe's Morphol. Arb. 3, 1.
. 1899. Zur Entwicklungsgeschichte des Rehes. Verh. Anat. Ges.,
Anat. Anz. Ergdnz. 16, 64.
. 1901. Frlihe Entwicklungsstudien des Rehes und die Gastrulation
der Siluger. Verh. Anat. Ges. Bonn, Anat. Anz. Ergdnz. 19, 184.
. 1902. Die Entwicklung des Rehes bis zur Anlage des Mesoblast.
Arch. Anat. u. Physiol,, Anat. Abt., 292.
Kingery, H. M. 1914. So-called parthenogenesis in the white mouse.
Biol. Bull. 27, 240.
, 1917. Oogenesis in the white mouse. /. Morphol. SO, 261.
Kingsbury, B. F. 1913. The morphogenesis of the mammalian ovary:
Felis domestica. Am. J. Anat. 15, 345.
. 1914. Interstitial cells of the mammalian ovary. Am. J. Anat. 16,
59.
KiRKHAM, W. B. 1915. The germ cell cycle in the mouse. Proc. Am.
Assoc. Anat., Anat. Rec. 10, 217.
142 THE EGGS OF MAMMALS
KiRKHAM, W. B. 1916. The prolonged gestation period in suckling mice.
Anat. Rec. 11, 31.
. 1918. Observations on the relation between suckling ai. le rate
of embryonic development in mice. J. Exp. Zool. 27, 49.
KoHNO, S. 1925. Zur Kenntnis der Keimbahn des Menschen. Arch.
Gyjiak. 126, 310.
KuscHAKEWiTscH, S. 1910. Die Entwicklungsgeschichte der Keim-
drusen von Rana esculenta. Festschr. f. R. Hertwig 2, 61.
Krasovskaja, 0. V. 1935a. Fertilization of the rabbit egg outside the
organism. Contribution Z'^. Variations of size of the rabbit eggs
before and after fertilization (in Russian). Biol. Jour. 4, 262.
. 19356. Cytological study of the heterogeneous fertilization of the
egg of the rabbit outside the organism. Acta Zoologica 16, 449.
Kynoch, J. A. C. 1902. Repeated ovariotomy. /. Obst. and Gynec. Brit.
Emp. 2, 366.
Lams, H. 1910. Recherches sur I'oeuf de cobaye (Cavia cobaya), matura-
tion, fecondation, segmentation. Compt. rend. Assoc. Anat. Bruxelles
12, 119.
. 1913. fitude de I'oeuf de cobaye aux premiers stades de Fembryo-
genese. Arch. Biol. 28, 229.
. 1924. L'oeuf de la rate pendant les premieres phases de son de-
veloppement avant son arrivee dans Futerus. Compt. rend. Assoc.
Anat. Bruxelles, 195.
Lams, H. and Doorme, J. 1908. Nouvelles recherches sur la maturation
et la fecondation de Foeuf des mammiferes. Arch. Biol. 23, 259.
Lane-Claypon, J. E. 1905. On the origin and life history of the inter-
stitial cells of the ovary of the rabbit. Proc. Roy. Soc. B 77 ^ 32.
. 1907. On ovogenesis and the formation of the interstitial cells of
the ovary. /. Ohst. and Gynec. 11, 205.
Lane, C. E. 1935. Some influences of oestrin on the hypophyseal-gonad
complex of the immature female rat. Am. J. Physiol 110, 681.
Lane, C. E. and Hisaw, F. L. 1934. The follicular apparatus of the ovary
of the immature rat and some factors which influence it. Anat. Rec.
60 Supp. 52.
Lange, J. 1896. Die Bildung der Eier und der Graafschen Follikel bei
der Maus. Verb. phys. med. Gesell. Wurzburg N. F. 30, 57.
League, B. and Hartman, C. G. 1925. Anovular Graafian follicles in
mammalian ovaries. Anat. Rec. 30, 1.
Lee, F. C. 1928. The tubo-uterine junction in various mammals. Bull.
Johns Hopkins Hosp. 42, 335.
BIBLIOGRAPHY 143
Lelievre, Peyron et Corsy. 1927. La parthenogenese dans I'ovaire
des mammiferes et le probleme de I'origine des embryonies. Bull.
pf etude de Cancer 16, 71L
Leonard, S. L., Hisaw, F. L. and Fevold, H. L. 193L Further studies
of the folHcular-corpus luteum hormone relationship in the rabbit.
Am. J. Physiol. 100, UL
Leonard, S. L., Meyer, R. K. and Hisaw, F. L. 193 L The effects of
oestrin on the development of the ovary in immature female rats.
Endocrinology IS, 17.
Lewis, L. L. 1911. The vitality of reproductive cells. Okla. Agr. Exp.
Sta, Bull. 96.
Lewis, W. H. 1931. Living mouse eggs. Anat. Rec. 48, 52.
Lewis, W. H. and Gregory, P. W. 1929. Cinematographs of living
developing rabbit eggs. Science 69, 226.
Lewis, W. H. and Hartman, C. G. 1933. Early cleavage stages of the
egg of the monkey. Carnegie Inst. Wash. Contrib. Embryol. 24, 187.
Lewis, W. H. and Wright, E. S. 1935. On the early development of the
mouse egg. Carnegie Inst. Wash. Contrib. Embryol. 25, 113.
LiLLiE, F. 1919. Problems of fertilization. University of Chicago Press.
Chicago.
LiLLiE, R. S. 1934. The influence of hypertonic and hypotonic sea water
on artificial activation of starfish eggs. Biol. Bull. 66, 361.
LiPSCHtJTZ, A. 1924. The internal secretions of the sex glands. Williams
and Wilkins. Baltimore.
. 1928. New developments in ovarian dynamics and the law of
follicular constancy. Brit. J. Exp. Biol. 5, 283.
LiPSCHUTz, A., Kallas, H. and Pabz, R. 1929. Hypophyse und Gesetz
der Pubertat. Arch. ges. Physiol. 221, 695.
LiPSCHUTz, A. and Voss, H. E. 1925. Further developments on the dy-
namics of ovarian hypertrophy. Brit. J. Exp. Biol. 3, 35.
LoEB, J. 1895. Untersuchungen iiber die physiologischen Wirkungen des
Sauerstoffmangels. Arch. ges. Physiol. 62, 249.
. 1906. Versuchen iiber den Qhemischen Charakter des Befruch-
tungsvorgangs. Biochem. Z. 1, 183.
. 1913. Artificial parthenogenesis and fertilization. University of
Chicago Press. Chicago.
LoEB, J. and Wastenys, H. 1912. Die Oxydationsvorgange im befruch-
teten und unbefruchteten Seesternei. Arch. Entwcklngsmech. 35, 555.
LoEB, L. 1901. On progressive changes in the ova in mammalian ovaries.
J. Med. Res. 6, 39.
144 THE EGGS OF MAMMALS
LoEB, L. 1905. Uber hypertropische Vorgange bei den Follikelatresie
nebst Bemerkungen liber die oocyten in den Markstrangen und liber
Teilungserscheinungen am Ei im ovarium des Meerschweinchen.
Arch. mikr. Anat. 65. 728.
. 1911a. Beitrage zur Analyse des Gewebewachstums. VII. Uber
einige Bedingungen des Wachstums der embryonalen Placenta. Arch.
Entwcklngsmech. 32, 662.
. 19116. The parthenogenetic development of ova in the mammalian
ovary and the origin of ovarian teratomata and chorioepitheliomata.
/. Am. Med. Assoc. 56, 1327.
. 1912. Uber chorionepitheliomartige Gebilde im Ovariums des
Meerschweinchens und liber ihre warseheinliche Entstehung aus
parthenogenetisch sich entwickelnden Eiern. Z. Krebsforsch. 11,1.
. 1915. An early stage and an experimentally produced extrauterine
pregnancy and the spontaneous parthenogenesis of the eggs in the
ovary of the guinea pig. Biol. Bull. 28, 59.
. 1917. Factors in the growth and sterility of the mammahan ovary.
Science JfS, 591.
. 1923. The parthenogenetic development of eggs in the ovary of
the guinea pig. Science 58, 35.
. 1932. The parthenogenetic development of eggs in the ovary of
the guinea pig. Anat. Rec. 51, 373.
Long, J. A. 1912. Studies on early stages of development in rats and
mice. Univ. Calif. Pub. Zool. 6, 105.
Long, J. A. and Evans, H. M. 1922. The oestrus cycle in the rat and
associated phenomena. Mem. Univ. Calif. 6, 1.
Long, J. A. and Mark, E. L. 1911. The maturation of the egg of the
mouse. Carnegie Inst. Wash. Publ. 1^2, 1.
Lowenthal, N. 1888. Zur Kenntnis des Keimfieckes im Urei einiger
Sanger. Anat. Am. 3, 363.
Lyon, E. P. 1902. Effects of KCN and of lack of O2 upon the fertilized
eggs and the embryos of the sea-urchin. Am. J. Physiol. 7, 56.
MacDowell, E. C, Allen, E. and MacDowell, C. G. 1929. The rela-
tion of parity, age, and body weight to the number of corpora lutea
in mice. Anat. Rec. 4I, 267.
MacDowell, E. C. and Lord, E. M. 1925. The number of corpora lutea
in successive pregnancies. Anat. Rec. 31, 131.
Mann, M. C. 1924. Cytological changes in unfertilized tubal eggs of the
rat. Biol. Bull. 46, 316.
BIBLIOGRAPHY 145
Marshak, a. 1935. The effect of x-rays on chromosomes in different
stages of meiosis. /. Gen. Physiol. 19, 179.
Marshall, F. H. A. and Jolly, W. A. 1907. Results of removal and
transplantation of ovaries. Trans. Roy. Soc. Edinburgh 4^, 589.
. 1908. On the results of heteroplastic transplantation as compared
with those produced by transplantation in the same individual.
Quar. J. Exp. Physiol. 1, 115.
Melissinos, K. 1907. Die Entwicklung des Eies der Mause von den
ersten Furchung-Phanomenen bis zur Festsetzung der Allantois an
der Etoplacentasplatte. Arch, niikr. Anal. 70, 577.
Meredith, W. A. 1904. Pregnancy after removal of both ovaries for
dermoid tumor. Brit. Med. J. 1, 1360.
Meyer, R. 1913. Ueber die Beziehung der Eizelle und des befruchteter
Eies zum FoUikelapparat, sowie des Corpus luteum zur menstruation.
Arch. Gyndk. 100, 1.
Meyer, R. K., Leonard, S. L., Hisaw, F. L. and Martin, S. J. 1932.
The influence of oestrin on the gonad-stimulating complex of the
anterior pituitary of castrated male and female rats. Endocrinology 16,
655.
Meyerhof, 0. und Kiessling, W. 1933. Uber das Auftreten und den
Umsatz der a-glycerinphosphorsaure bei der Enzymatischen Kohlen-
hydratspaltung. Biochem. Z. 264, 40.
Minot, C. S. 1889. Segmentation of the ovum with special reference to
the Manomalia. Am. Nat. 23, 463.
Morris, M 1917. A cytological study of artificial parthenogenesis in
Cumingia. /. Exp. Zool. 22, 1.
Morris, M. M. 1901. Pregnancy following removal of both ovaries and
tubes. Boston Med. and Surg. J. 144, 86.
McPhail, M. K. 1933. Studies on the hypophysectomized ferret. Proc.
Roy. Soc. B 114, 124.
Needham, J. 1932. Chemical embryology. Cambridge University Press.
Cambridge.
Nelson, W. 0., Pfiffner, J. J. and Haterius, H. 0. 1930. The pro-
longation of pregnancy by extracts of corpus luteum. Am. J. Physiol.
91, 690.
Newman, H. H. 1912. The ovum of the nine-banded armadillo:
growth of the ovocytes, maturation, and fertilization. Biol. Bull.
23, 100.
. 1913. Parthenogenetic cleavage of the armadillo ovum. Biol.
Bull. 25, 54.
146 THE EGGS OF MAMMALS
Nicholas, J. S. 1933a. Development of transplanted rat eggs. Proc.
Soc. Exp. Biol. & Med. 30,1111.
. 19336. The development of rat embryonic tissues after transplanta-
tion of the egg to the kidney. Anat. Rec. 55, Supp. 31.
. 1934. Experiments on developing rats. I. Limits of foetal regenera-
tion; behavior of embryonic material in abnormal environments.
Anat. Rec. 58, 387.
Nicholas, J. S. and Rudnick, D. 1933. The development of embryonic
rat tissues upon the chick chorioallantois. J. Exp. Zool. 66, 193.
. 1934. The development of rat embryos in tissue culture. Proc.
Nat. Acad. Sc. 20, 656.
Novak, J. and Eisinger, K. 1923. Ueber kunstlick bewirkte Teilung
des unbefruchteten Saugertiereies. Arch. mikr. Anat. 98, 10.
NussBAUM, M. 1880. Zur Differenzierung des Geschlechts im Thierreich.
Arch. mikr. Anat. 18, 1.
VAN OoRDT, G. J. 1921. Early developmental stages of Manis javanica
Desm. Verhandl. kon. Scad, ven Wetensch. Amsterdam, Sec. 2, Part
XXI, 1.
Palladino, G. 1887. Ulteriori ricerche sulla distruzione e rinovamento
continuo del parenchima ovarico dei Mammiferi. Tipi del cav.
Antonio Morano. Napoli.
. 1894. La destruction et le renouvellement continuel du parenchyme
ovarique des mammiferes. Arch. ital. Biol. 21, XV Sec. d'Anat.
. 1898. Sur le type du structure de I'ovariere. Arch. ital. Biol. 29, 139.
Pallot, G. 1928. A propos de la regeneration ovarienne et des modifica-
tions periodiques de I'epithelium vaginal chez le rat blanc. Compt.
rend. Soc. biol. 99, 1333.
Papanicolou, G. N. 1925. Ovogenesis during sexual maturity as eluci-
dated by experimental methods. Proc. Soc. Exp. Biol. & Med. 21, 393.
Parker, G. H. 1931. The passage of sperms and eggs through the ovi-
ducts in terrestrial vertebrates. Phil. Trans. Roy. Soc. London B 219,
381.
Parkes, a. S. 1926. On the occurrence of the oestrus cycle after x-ray
sterilization. Part I. Irradiation of mice at three weeks old. Proc.
Roy. Soc. B 100, 172.
. 1927a. On the occurrence of the oestrus cycle after x-ray steriliza-
tion. Part 11. Irradiation at or before birth. Proc. Roy. Soc. B 101, 71.
. 19276. On the occurrence of the oestrus cycle after x-ray steriliza-
tion. Part III. The periodicity of oestrus after sterilization of the
adult. Proc. Roy. Soc. B 101, 421.
BIBLIOGRAPHY 147
Parkes, a. S. 1927c. On the occurrence of the oestrus cycle after x-ray
sterilization. Part IV. Irradiation of the adult during pregnancy and
lactation; and general summary. Proc. Roy. Soc. B 102, 51.
. 1929 The internal secretions of the ovary. Longmans, Green and
Co. London.
. 1931. The reproductive processes of certain mammals. II. The
size of the Graafian follicle at ovulation. Proc. Roy. Soc. B 109,
185.
Parkes, A. S., Fielding, Una and Brambell, F. W. R. 1927. Ovarian
regeneration in the mouse after complete double ovariotomy. Proc.
Roy. Soc. B 101, 328.
Parkes, A. S., Rowlands, I. W. and Brambell, F. W. R. 1932. Effects
of x-ray sterilization on oestrus in the ferret. Proc. Roy. Soc. B 109,
425.
Pearl, R. and Schoppe, W. F. 1921. Studies on the physiology of repro-
duction in the domestic fowl. /. Exp. Zool. 34, 101.
Pencharz, Richard. 1929. Experiments concerning ovarian regeneration
in the white rat and white mouse. /. Exp. Zool. 54, 319.
PpLtJGER, E. 1863. Die Eierstocke der Saugetiere und der Menschen.
Wilhelm Engelmann. Leipzig.
PiNCUS, G. 1930. Observations on the living eggs of the rabbit. Proc.
Roy. Soc. 107, 132.
. 1931. The transplantation of mouse ovaries into the rat. Anat.
Rec. 49, 97.
PiNCUS, G. and Enzmann, E. V. 1932. Fertilization in the rabbit. /. Exp.
Biol 9, 403.
. 1934. Can mammalian eggs undergo normal development in vitro?
Proc. Nat. Acad. Sc. 20, 121,
. 1935. The comparative behavior of mammalian eggs in vivo and
in vitro. I. The activation of ovarian eggs. /. Exp. Med. 62, 665.
. 1936a. The comparative behavior of mammalian eggs in vivo and
in vitro. II. The activation of tubal eggs of the rabbit. J. Exp.
Zool. 73, 195.
. 19366. The growth, maturation and atresia of the ovarian eggs of
the rabbit. (In press.)
PiNCUS, G. and Kirsch, R. E. 1936. The sterility in rabbits produced
by injections of oestrone and related compounds. Am. J. Physiol.
115, 219.
PiNcus, G. and Werthessen, N. 1933. The continued injection of oestrin
into young rats. Am. J. Physiol. 103, 631.
148 THE EGGS OF MAMMALS
QUINLA.N, J., Mare, G. S. and Roux, L. L. 1932. 18th Rep. Div. Vet.
Serv. and Anim. Ind. Union of South Africa, Pt. II, 813.
Rabl, H. 1898. Zur Kenntnis der RichtungsspindeLn in degenerierenden
Saugetiereiern. Sitzber. Akad. Wiss. Naturiciss. Kl. 106, 95.
Reagan, F. P. 1916. Some results and possibilities of early embryonic
castration. Anat. Rec. 11, 489.
Reichert, K. 1861. Beitrage zur Entwicklungsgeschichte des Meer-
schweinchens. Abhandl. K. Akad. Wissensch. 182. Berhn.
Rein, G. 1883. Beitrage zur Kentniss der Reifungserscheinungen und
Befruchtungsvorgange am Saugethierei. Arch. mikr. Anat. 22,
233.
Reiss, M., Selye, H. und Balint, J. 1931a. Uber die Wirkung alkahscher
Hypophysenvorderlappenextrakte auf das Genitale der weibhchen
Ratte. Endokrinol. 8, 15.
. 19316. tjber die Beeinflussung des mannhchen Genitales durch den
luteinisierenden Wirkstoff des Hypophysenvorderlappens. Endo-
krinol. 5, 81.
Robertson, J. A. 1890. Renewal of menstruation and subsequent preg-
nancy after removal of both ovaries. Brit. Med. J. 2, 722.
Robinson, A. 1892. Observations upon the development of the segmenta-
tion cavity, the archenteron, the germinal layers, and the amnion
in mammals. Quar. J. Micr. Sc. 33, 369.
. 1904. Lectures on the early stages in the development of mamma-
lian ova and on the formation of the placenta in different groups of
mammals. /. Anat. 38, 186.
. 1918. The formation, rupture and closure of ovarian follicles in
ferrets and ferret-polecat hybrids, and some associated phenomena.
Trans. Roy. Soc. Edinburgh 52, 303.
RuBASCHKiN, W. 1906. Uber die Veranderungen der Eier in den zugrunde-
gehenden Graafschen Follikeln. Aimt. Heft£ 32, 255.
. 1908. Zur frage von der Ent-stehung der Keimzellen bei Sauge-
tierembryonen. Anat. Anz. 32, 222.
. 1910. Uber das erste Auftreten und Migration der Keimzellen bei
Saugetierembryonen. Anat. Hefte J^l, 243.
. 1912. Zur lehre von der Keimbahn bei Saugetieren. Uber die
Entwicklung der Keimdriisen. Anat. Hefte Jj6, 342.
RuNNSTROM, J. 1930. Atmungsmechanismus und entwicklungserregung
bei dem Seeigelei. Protopla^ma 10, 106.
. 1933. Zur Kenntnis des Stoffwechselvorgange bei der Entwick-
lungserregung des Seeigeleies. Biochem. Z. 258, 257.
BIBLIOGRAPHY 149
RuNNSTROM, J. 1935. On the influence of pyocyanine on the respiration
of the sea urchin egg. Biol. Bull. 68, 327.
Sainmont, G. 1905. Recherches relatives a I'organogenese du testicle
et de I'ovaire chez le chat. Arch. Biol. 22, 71.
Sakurai, T. 1906. Normentafel zur Entwicklungsgeschichte des Rehes
(Cervus capreolus). Verlag von Gustav Fischer. Jena.
Sansom, G. S. 1920. Parthenogenesis in the water vole. /. Anal. 55,
68.
Sansom, G. S. and Hill, J. P. 1930. Observations on the structure and
mode of implantation of the blastocyst of Cavia. Tram. Zool. Soc.
London 21, 295.
Schottlander, G. 1891. Beitrag zur Kenntnis der Follikelatresie nebst
einigen Bemerkungen liber die unveriinderten Follikel in den Eier-
stocken der Saugetiere. Arch. mikr. Anal. 37, 192.
ScHRON, 0. 1863. Beitrag zur Kentniss der Anatomie und Physiologic
des Eierstockes der Siiugethiere. Z. ivissensch. Zool. 12, 409.
ScHULTZ, W. 1900. Transplantation der Ovarien auf miinnliche Tiere.
Zenlr. allg. Path. 2, 200.
Scott, J. 1906. Morphology of the parthenogenetic development of
Amphitrite. /. Exp. Zool. 3, 49.
Selenka, E. 1883. Studien fiber die Entwicklungsgeschichte der Thiere.
1 Heft, Keimbliitter und Primitivorgane der Maus. C. W. Kreidel's
Verlag. Wiesbaden.
. 1884. Studien fiber die Entwicklungsgeschichte der Thiere. 3 Heft,
Die Blatterumkehrung im Ei der Nagethiere. C. W. Kreidel's Verlag.
Wiesbaden.
. 1887. Studien fiber die Entwicklungsgeschichte der Thiere. 4 Heft.
Das Opossum. C. W. Kreidel's Verlag. Wiesbaden.
Selye, H. 1933. Effect of hypophysectomy on the ovary of immature
rats. Proc. Soc. E.vp. Biol. & Med. 31, 262.
Selye, H. and Collip, J. B. 1933. Production of exclusively thecal
luteinization and continuous oestrus with anterior-pituitary-liko hor-
mone. Proc. Soc. Exp. Biol. & Med. 30, 647.
Selye, H., Collip, J. B. and Thomson, D. L. 1935^7. Endocrine inter-
relationships during pregnancy. Endocrinology 19, 151.
. 1935/;. Effect of oestrin on ovaries and adrenals. Proc. Soc. Exp.
Biol. & Med. 32, 1377.
Selye, H. and McKeown, T. 1934a. Production of pseudopregnancy by
mechanical stimulation of the nipples. Proc. Soc. Exp. Biol. c(' Med.
31, 683.
150 THE EGGS OF MAMMALS
Selye, H. and McKeown, T. 19346. Further studies on the influence of
suckhng. Anat. Rec. 60, 323.
SiMKiNS, C. S. 1923. Origin and migration of the so-called primordial
germ cells in the mouse and rat. Acta Zool. 4, 241.
. 1928. Origin of sex cells in man. Am. J. Anat. p, 249.
Slawinsky, K. 1873. Filaments glandulaires trouves dans Tovaire d'une
femme adulte. Bull. Soc. Anat. Paris 4-8, 844.
Slonaker, J. R. 1927. Semi-ovariectomy, compensatory hypertrophy
of the remaining ovary and migration of the ova in the albino rat.
Am. J. Physiol. 81, 620.
Smith, P. E. 1930. Hypophysectomy and a replacement therapy in the
rat. Am. J. Anat. 1^.5, 205.
. 1932. The effect on the reproductive system of ablation and im-
plantation of the anterior hypophysis. In Allen: Sex and Internal
Secretions, 734. Williams and Wilkins. Baltimore.
Smith, P. E. and Engle, E. T. 1927. Experimental evidence regarding
the role of the anterior pituitary in the development and regulation
of the genital system. Am. J. Anat. Jfi, 159.
Smith, P. E. and MacDowell, E. C. 1931. The differential effect of
hereditary mouse dwarfism on the anterior-pituitary hormones. Anai.
Rec. 50, 85.
Smith, P. E. and White, W. E. 1931. The effect of hypophysectomy on
ovulation and corpus luteum formation in the rabbit. /. Am. Med.
Assoc. 97, 1861.
Smith, S. C. 1925. Degenerative changes in the unfertilized uterine eggs
of the opossum (Didelphis virginiana), with remarks on the so-called
parthenogenesis in mammals. Am. J. Anat. 35, 81.
Snyder, F. F. 1923. Changes in the fallopian tube during the ovula-
tion cycle and early pregnancy. Bull. Johns Hopkins Hosp. 34-,
121.
. 1934. The prolongation of pregnancy and complications of parturi-
tion in the rabbit following induction of ovulation near term. Bull.
Johns Hopkins Hosp. 54, 1.
SoBOTTA, J. 1893. Mitteilungen liber die Vorgange bei der Reifung,
Befruchtung und erste Furchung des Eies der Maus. Verhandl.
Anat. Ges. Gottingen, 111.
. 1895. Die Befruchtung und Furchung des Eies der Maus. Arch.
mikr. Anat. 45, 15.
. 1899. Uber die Bedeutung der mitotischen Figuren in den Eier-
stockseiern der Saugetieren. Wurzburg.
BIBLIOGRAPHY 151
SoBOTTA, J. und BuRCKHARD, G. 1911. Rcifung und Befruchtung des
Eies der weissen Ratte. Anat. Hefte 42, 433.
Spee, F. 1915. Anatomie und Physiologie der Schwangerschaft. 1. Theil
in Doderlein: Handbuch der Geburtshilfe 1. Bergmann. Munich.
Spencer, J., D'Amour, F. E. and Gustavson, R. G. 1932. Effects of
continued oestrin injections on young rats. Am. J. Anat. 50, 129.
Spuler, a. 1900. Uber die Teilungserscheinungen der Eizellen in degen-
erierenden Follikeln des Saugerovariums. Anat. Hefte 16, 85.
Squier, R. R. 1932. The living egg and early stages of its development
in the guinea-pig. Carnegie Inst. Wash. Contrih. Emhnjol. 23, 225.
Stockard, C. R. and Papanicolou, G. N. 1917. The existence of a
typical oestrus cycle in the guinea-pig, with a study of its histological
and physiological changes. Am. J. Anat. 22, 225.
Stotsenburg, J. M. 1913. The effect of spaying and semi-spaying young
albino rats (M. norvegicus albinus) on the growth in body weight and
body length. Anat. Rec. 7, 183.
Streeter, G. L. 1931. Development of the egg as seen by the embry-
ologist. Sc. Month. 32, 495.
Sutton, R. S. 1896. Double ovariotomy followed by pregnancy and de-
livery at term. Trans. Am. Gynec. Soc. 21, 109.
SwANN, W. F. G. and del Rosario, C. 1932. The effect of certain mono-
chromatic ultra-violet radiation upon Euglena cells. J. Franklin
Inst. 213, 549.
SwEZY, 0. 1929. The ovarian chromosome cycle in a mixed rat strain.
J. Morphol. & Physiol. 48, 445.
. 1933a. The changing concept of ovarian rhythms. Quar. Rev.
Biol. 8, 423.
. 19336. Ovogenesis and the hypophysis: the effects of pregnancy,
hypophysectomy, thyroidectomy and hormone administration on the
ovary of the rat. Science Press. Lancaster.
SwEZY, 0. and Evans, H. M. 1930. Ovarian changes during pregnancy in
the rat. Science 71, 46.
Tafani, a. 1889. I primi momenti delb sviluppo dei mammiferi. Rendi-
conti R. Accad. Lincei 5, 119.
Tamura, Y 1926. The effects of implantation upon ovarian grafts in the
male mouse. Proc. Roy. Soc. Edinburgh 47, 148.
Teel, H. M. 1926. The effect of injecting anterior hypophysial fluid on
the course of gestation in the rat. Am. J. Physiol. 79, 120.
Turner, C. W. 1932. The mammary glands. In Allen: Sex and Internal
Secretions. Williams and Wilkins. Baltimore.
152 THE EGGS OF MAMMALS
Van Beneden, E. 1875. La maturation de I'oeuf, la fecondation et les
premieres phases du developpement embryonaire des Mammiferes
d'apres les recherches faites sur le Lapin. Bull. Acad. Roy. Sc. Lettres
et Beaux Arts Belg. J^O, 686.
. 1880. Contribution a la connaissance de I'ovaire des mammiferes.
Arch. Biol. 1, 475.
. 1899. Recherches sur les premiers stades du developpement du
Murin (Vespertilio murinus). Anat. Anz. 16, 307.
. 1911. De la segmentation, de la formation de la cavite blasto-
dermique et de I'embryon didermique chez le Murin. Arch. Biol.
26, 1.
. 1912. Recherches sur Fembryologie des mammiferes. Arch. Biol.
27, 191.
Van Beneden, E. et Julin, C. 1880. Observations sur la maturation,
la fecondation et la segmentation de I'oeuf chez les Cheiropteres.
Arch. Biol. ^ 551.
Van der Stricht, 0. 1901. L'atresie ovulaire et I'atresie foUiculaire du
follicule de De Graaf dans Fovaire de chauve-souris. Verhandl. anat.
Ges. 15, Versamml. Bonn.
Vanneman, a. S. 1917. The early history of the germ cells in the armadillo
Tatusia novemcineta. Am. J. Anat. 22, 341.
Voss, H. E. 1925. Condition de la greffe ovarienne intratesticulaire.
Compt. rend. Soc. biol. 93, 1066.
Waddington, C. H. and Waterman, A. J. 1933. The development in
vitro of young rabbit embryos. /. Anat. 67, 356.
Wagener, G. 1879. Bemerkungen iiber den Eierstock und den Gelben
Korper. Arch. Anat. u. Physiol. 175.
Wagner, R. 1836. Prodromus historiae generationis hominis atque
animahum. Leipzig.
Waldeyer, W. 1870. Eierstock und Ei. Verlag von Wilhelm Engelmann.
Leipzig.
. 1906. Die Geschlechtzellen. In: Handbuch der vergleichenden
und experimentellen Entwickelunglehre der Wirbeltiere. Oskar Hert-
\vig. Jena.
Walsh, L. S. M. 1917. The growth of the ovarian follicle in the guinea
pig under normal and pathological conditions. J. Exp. Med. 26, 245.
Walton, A. and Hammond, J. 1932. Observations on ovulation in the
rabbit. Brit. J. Exp. Biol. 6, 190.
Wang, G. H. and Guttmacher, A. F. 1927. The effect of ovarian trau-
matization on the spontaneous activity and genital tract of the albino
BIBLIOGRAPHY 153
rat, correlated with a histological study of the ovaries. Ayn. J. Physiol.
82, 335.
Warburg, O. 1908. Beobachtungen fiber die Oxydationsprocesse im
Seeigelei. Z. physiol. C/iem. 57, 1.
. 1909. Ue})er die Oxydation im Ei: II, Mittheilung. Z. physiol.
Chem. 60, 443.
. 1910. Ueber die Oxydationen in lebenden Zellen nach Versuchen
am Seeigelei. Z. physiol. Chem. 66, 305.
. 1914rt. Uel)er die Rolle des Eisens in der Atmung des Seeigeleis
nebst Bermerkungen iiber einige durch Eisen beschleunigte Oxyda-
tionen. Z. physiol. Chem. 92, 231.
. 19146. Zellstruktur und Oxydationsgeschwindigkeit nach Ver-
suchen am Seeigelei. Pflug. Arch. ges. Physiol. 158, 189.
. 1932. Das sauerstoffiibertragende Ferment der Atmung. Z. angew.
Chem. Jf.5, 1.
Waterman, A. J. 1932. Culture * in vitro ' and transplantation of young
rabbit embryos. Anat. Rec. 54, 72.
. 1934. Survival of young rabbit embryos on artificial media. Proc.
Nat. Acad. Sc. 20, 145.
Weil, C. 1873. Beitrage zur Kentniss der Befruchtung und Entwicklung
des Kanincheneies. Strieker's med. Jahrb.
Weismann, August. 1883. Enstehung der Sexuallzellen bei den Hydro-
medusae. Verlag von Gustav Fischer. Jena.
. 1904. Vortrage liber Descendenztheorie. English translation.
Edward Arnold. London.
Whitaker, D. M. 1933. On the rate of oxygen consumption by fertilized
and unfertilized eggs. V. Comparisons and interpretation. /. Gen.
Physiol. 16, 497.
Williams, W. L. 1909. Veterinary obstetrics including the diseases of
the breeding animals and the new-born. Williams. Ithaca.
WiLLiER, B. H. 1921. Structures and homologies of free-martin gonads.
/. Exp. Zool. 33, 63.
. 1932. The embryological foundations of sex in vertebrates. In
Allen: Sex and Internal Secretions. Williams and Wilkins. Balti-
more.
. 1933a. Potencies of the gonad-forming area in the chick as tested
in chorio-allantoic grafts. Arch. Entwcklngsmech. Organ. 130, 616.
. 19336. On the origin and differentiation of the sexual gland. Am.
Nat. 67, 1.
Wilson, E. B. 1901. Experimental studies in cytology. I. A cytological
154 THE EGGS OF MAMMALS
study of artificial parthenogenesis in sea urchin eggs. Arch. Ent-
wcklngsmech. 12, 529.
Wilson, J. T. 1928. On the question of the interpretation of the struc-
tural features of the early blastocyst of the guinea-pig. /. Anat. 62,
346.
Wilson, J. T. and Hill, J. P. 1907. Observations on the development of
Ornithorhynchus. Phil. Trans. Roy. Soc. 199 B, 31.
DE Winiwarter, H. 1901. Recherches sur Tovogenese et I'organogenese
de Fovaire des mammiferes (lapin et homme). Arch. Biol. 17, 33.
. 1910. Contributions a I'etude de Fovaire humaine. I. Appareil
nerveux et pheochrome. II. Tissue musculaire. III. Cordons medul-
laires et corticaux. Arch. Biol. 25, 683.
. 1920. Couche corticale definitive au hile de I'ovaire et pseudo-
neoformation ovulaire. Compt. rend. Soc. biol. 83, 1406.
DE Winiwarter, H. et Sainmont, G. 1909. Nouvelles recherches sur
Tovogenese et I'organogenese de Fovaire des mammiferes (chat).
Arch. Biol. 24, 1.
WiSLOCKi, G. B. and Goodman, L. 1934. The effect of anterior lobe
extract or concentrated human urine of pregnancy upon the early
part of gestation in the rabbit. Anat. Rec. 69, 375.
WiSLOCKi, G. B. and Snyder, F. F. 1931. On the experimental production
of superfetation. Bull. Johns Hopkins Hasp. 4-9, 106.
WiTscHi, E. 1929. Studies on sex differentiation and sex determination
in amphibians. II. Sex reversal in female tadpoles of Rana sylvatica
following the application of high temperature. /. Exp. Zool. 52, 267.
Yamane, J. 1930. The proteolytic action of mammalian spermatozoa
and its bearing upon the second maturation division of ova. Cytologia
1, 394
. 1935. Kausal-analytische Studien iiber die Befruchtung des Kanin-
cheneies. I. Die Dispersion der Follikelzellen und die Ablosung der
Zellen der Corona radiata des Eies durch Spermatozoen. Cytologia 6,
233.
Zondek, B. 1931. Die Hormone des Ovariums und des Hypophysen-
vorderlappens. Julius Springer. Berlin.
AUTHOR INDEX
Addison, W. H. F., 52
Allen, B. M., 30
Allen, E., 8, 9, 10, 14, 19, 42, 44, 45,
57, 63, 71
Allen, W. M., 117
Amann, J. A., 9
Anderson, D. H., 74
Aral, H., 8, 19, 23, 24, 32, 33, 39
Asami, G., 44
Aschner, B., 7
Asdell, S. A., 18, 86
Assheton, R., 112
Athias, M., 21, 52
von Baer, K. E., 1
Balfour, F. M., 8, 52
Balint, J., 26
Barry, M., 1, 2, 62
Bellerby, C. W., 48
Benoit, J., 29
Biedl, A., 96
Bischoff, T. L. W., 2, 62, 124
Bland, L. J., 57, 63, 71
Bond, C. S., 18
Bonnet, R., 2, 52, 124
Boone, C, 18
Bosaeus, W., 53
Brachet, A., 3, 112, 121
Brambell, F. W. R., 6, 7, 11, 16, 21,
30, 32, 34, 36
Branca, A., 53, 54
Buhler, A., 8
Burckhard, G., 82
Burdick, H. O., 94, 116, 117
Butcher, Earl O., 8, 20
Buyse, Adrian, 31
Caldwell, W. H., 2
Campbell, J. A., 95
Carmichael, E. S., 18
Carrel, A., 55
Casida, L. E., 33
Castle, W. E., 89, 92, 93
Champy, C, 98
Chapin, Catherine L., 30
Charlton, H. H., 73
Clark, E. B., 52
Clark, R. T., 68
Coert, H. J., 9
Cole, H. H., 51, 71
Collier, W. D., 26, 27
Collip, J. B., 33, 115, 126
Corey, E. L., 33
Corner, G. W., 94, 116, 117
Corsey, 53
Courrier, R., 53, 61, 123
Cowperthwaite, Marian M., 7, 10
Crew, F. A. E., 18
Cruikshank, W., 62, 112
Curtis, J., 26, 27
Dalq, A., 110
D'Amour, F. E., 26
Davenport, C. B., 15, 16
Deansley, R., 48
Defrise, A., 3, 62, 65
Del Rosario, C, 110
Doisy, E. A., 26, 27
Domm, L. V., 29
Doorme, J., 2, 82
Doran, M. A., 18
Eisinger, K., 2, 111
Emery, F. E., 18, 19
Engle, E. T., 24, 26, 33, 35, 42, 43, 44,
45, 51, 52, 53
Enzmann, E. V., 36, 38, 44, 46, 50, 56,
66, 67, 75, 76, 78, 81, 93, 95, 96, 97,
108, 110, 111, 116, 123, 124
Evans, H. M., 2, 8, 11, 14, 19, 23, 24,
26, 44, 51, 52, 71, 88, 125
Fee, A. R., 48
Fehx, W., 7, 31
Fellner, O. O., 9
Fevold, H. L., 27, 123
Fielding, Una, 16, 21
Firket, J., 8
Fischer, Albert, 55
Flemming, W., 53
Foster, M. A., 27
Foulis, J., 8
155
156
AUTHOR INDEX
Francis, B. F., 14, 19, 22
Friedgood, H., 49
Friedman, M. H., 48
Fuss, A., 6
Genther, I., 21
Gerard, P., 10
Gilchrist, F., 62, 71, 75, 81, 82,
89,94
Goodman, L., 126
Gordon, C. S., 18
Greep, O., 27
Gregory, P. W., 3, 65, 89, 91, 92, 93,
94, 112
Grosser, O., 124
Grusdew, W. S., 2, 53, 111
Gm'witsch, A., 53
Gustavson, R. G., 26
Gutherz, S., 55
Guttmacher, A. F., 19
Haberlandt, G., 55
Hiiggstrom, P., 53
Hall, B. v., 115
Hamlett, G. W. D., 126, 127
Hammond, J., 18, 33, 46, 82, 83, 84,
85, 86, 87
Hanson, F. B., 18
Hargitt, G. T., 8, 35
Harms, J. W., 5
Hartman, C. G., 2, 3, 18, 35, 40, 45,
65, 68, 72, 74, 87, 88, 89, 93
Hars^, W., 9
Hatai, S., 18
Haterius, H. O., 15, 16, 126
Heape, W., 2, 46
Hegner, R. W., 7
HcUbaum, A. A., 27
Hcnneguy, F., 53
Hensen, V., 2, 52, 74
Hertz, R., 33
Heuser, C. H, 89
Heys, Florence, 5, 16, 17, 18
Hill, J. P., 2, 11, 124
Hill, M., 2
Hindle, E., 103
Hinsey, J. C., 49
Hisaw, F. L., 26, 27, 28, 33, 54,
123
Hofstatler, R., 96
Huber, G. C., 2, 93, 95
Hubrecht, A. A. W., 2
Humphrey, R. R., 29
Janosik, J., 52
Jenkinson, J. W., 2, 6
Jolly, W. A., 20
Jones, T. W., 1
Julin, C., 2
Just, E. E., 54, 59
Kallas, H., 33
Kampmeier, O. F., 52
Kanel, V. Y., 18
Keibel, F., 2
Kiesling, W., 96
Kingery, H. M., 8, 52
Kingsbury, B. F., 8
Kirkham, W. B., 52, 124
Kirsch, R. E., 94, 95, 116, 117, 118,
119, 120, 122, 123
Kohno, S., 8
Kountz, W. B., 14, 19, 22
Krasovskaja, O. V., 80, 81
Kuschakewitsch, S., 29
Kynoch, J. A. C., 18
Lams, H., 2, 82
Lane, C. E., 27
Lane-Claypon, J. E., 9
Lange, J., 9
League, B., 35
Lee, F. C., 63
Lelievre, 53
Leonard, S. L., 27, 123
Lewis, L. L., 88
Lewis, W. H., 3, 62, 65, 66, 74, 88, 89,
93,94,112
Lillie, F., 54
LiUie, R. S., 54
Lipschutz, A., 18, 20, 33
Loeb, J., 54, 59, 95, 96, 108, 109
Loeb, L., 45, 53, 61
Long,J. A.,2, 52, 71,81,87, 88
Lord, E. M., 44
Lowenthal, N., 53
Lyon, E. P., 95
MacDowell, C. G., 44
MacDowell, E. C., 34, 44
McKeown, T., 127
McPhail, M. K., 48
Mann, M.C., 71,73, 88, 98, 111
Marc, G. S., 88
Mark, E. L., 87
Markee, J. E., 49
Marshak, A., 39
AUTHOR INDEX
157
MarshaU, F. H. A., 18, 20, 33, 82
Martin, S. J., 26
Melissinos, K., 2
Meredith, W. A., 18
Meyer, K., 26
Meyer, R., 45
Mever, R. K., 26
Meyerhof, O., 96
Minot, C. S., 2
Morris, M., 107
Morris, M. M., 18
Needham, J., 54, 95
XeLson, W. O., 126
Newell, Q. U., 57, 63, 71
Xewinan, H. H., 53
Nicholas, J. S., 3, 66, 67, 96, 114, 115
Novak, J., 2, 111
Xussbaum, M., 5, 6
Oberling, C, 53, 61
van Oordt, G. J. 2
Paez, R., 33
Palladino, G., 9, .53
Fallot, G., 16
Fapanicolou, G. X., 2, 8, 52
Farker, G. H., 74
Farkes, A. S., 16, 21, 33, 36, 38, 39,
46, 48, 127
Fearl R., 7
Fencharz, Richard, 16
Feters, H., 96
Fe\Ton, 53
Ff^ner, J. J., 126
Ffluger, E., 8, 53
Fincus, G., 15, 26, 36, 38, 44, 46, 49,
50, 56, 57, 62, 65, 66, 70, 71, 73, 75,
76, 78. 81, 82, 87, 89, 93, 94, 95, 96,
97, 98, 99, 100, 101, 102, 106, 108,
110, 111, 116, 117, 118, 119, 120,
121, 122, 123, 124
Fratt, J. R, 57, 63, 71
Quinlan, J., 88
Rabl, H., 53
Ra^^laud, R., 123
Reagan, F. F., 29
Reichert, K., 2
Rein, G., 2
Reiss, M., 26
Robertson- J. A., 18
Robinson, A., 2, 8, 47, 124
Roux, L. L., 88
Rowlands, I. W., 21
Rubaschkin, W., 7, 52
Rudnick, D., 3, 66, 114
Runnstrom, J., .54, 59, 95, 96
Sainmont, G., 6, 7, 30
Sakxirai, T., 2
Sansom, G. S., 53, 124
Saphir, X. R., 93, 124
Schoppe, W. F., 7
Schottlander, G., .53
Schron, O., 9 .
Schultz, W., 20
Scott, J., 107
Selenka, E., 2
Selye, H., 26, 32, 33, 115, 126, 127
Simkins, C. S., 8
Simon, S., 110
Simpson, M. E., 24, 26, 125
Slawinskv, K., 9
Slonaker, J. R., 18
Smith, F. E., 22, 24, 32, .33. .34, 44, 4.5,
48
Smith, S. C., 72
Snyder, F. F., .50, 93, 125
Sobotta, J., 2, .52, 82, 95
Spee, F., 62, 124
Spencer, J., 26
Spuler, A., .53
Squier, R. R., 3, 62, 66, 71, 89, 93, 94
Stockard, C. R., 2, .52
Stotsenburg, J. M., 18
Streeter, G. L., 41, 89
Subba Ran, A., 11
Sutton, R. S., 18
Swann, W. F. G., 110
Swezv, O., 8, 11, 14, 19, 22, 23, 24, 25,
26,27,32,44
Tafani, A., 2
Tamura, Y., 20, 21
Teel, H. M., 12.5, 126, 127
Thomson, D. L., 11-5, 126
Tribe, M., 2
TmTier, C. W., 127
Van Beneden, E., 2, 9, 94
Van der Stricht, O., .53
Vanneman, A. S., 7
Voss, H. E., 18, 20
Waddington, C. H., 113
Wagener, G., 9
158
AUTHOR INDEX
Wagner, R., 1
Waldeyer, W., 7
Walsh, L. S. M., 45
Walton, A., 46, 82, 87
Wang, G. H., 19
Warburg, O., 95, 96
Wastenys, H., 95
Waterman, A. J., 113
Weil, C, 2
Weismann, August, 5
Werthessen, N., 26
Whitaker, D. M., 54, 95
White, W. E., 48
Williams, W. L., 19
Wilher, B. H., 29, 30
Wilson, E. B., 107
Wilson, J. T., 2, 124
de Winiwarter, H., 6, 7, 8, 10
Wislocki, G. B., 50, 126
Witschi, E., 30
Wright, E. S., 3, 74, 89, 93
Yamane, J., 71, 75, 76, 78, 80
Zondek, B., 33
'«? /it
SUBJECT INDEX
Atresia, in follicles during oestrus
cycle and early pregnancy, 42, 43
in hypophysectomized rats, 32
prevention of, 44
Blastocyst, development in vitro,
112 and ff.
effect of ovariectomy on, 116
stages in the rabbit, 91
time of formation, 91, 93
Blastomeres, actual and prospe tive
in rabbit, 92, 93
transplantation of in rat, 97
Cleavage, and cyanide inhibition, 95,
96
and iodoacetate inhibition, 96
rate in various rabbit strains, 89
and ff.
rate of, 88 and ff.
relation to tubal and ovarian se-
cretions, 94 and ff.
stages in the rabbit, 90
Corpus luteum, and delayed im-
plantation, 126 and ff.
relation to blastocyst growth, 116
and ff.
Cortex, gonad, 30, 31
Deciduomata, and delayed preg-
nancy, 127
pH of, 116
Embryo, development in vitro, 113
and ff.
Endometrium, and ovum growth, 117,
123
Fertilization, effects of sperm dilu-
tion, 77, 78
relation to sperm extracts, 79
semination and ac \^a,tion, 75
and ff.
with irradiated sperm, 110
Follicle cells, formation, 10
Follicle graafian, antrum develop-
ment, 32, 37, 38
earliest response to pituitary hor-
mones, 33
growth in relation to ovum
growth, 34-39
in dwarf mice, 35
Follicle stimulating hormone and
ovogenesis, 26, 27
Follicles, anovular, 35
atresia during oestrus cycle and
early pregnancy, 42, 43
types in rabbits, 36, 37
Fragmentation, cinematography of,
105
Germ cells, meiosis, 7
origin, 5 and ff.
primordial, 6, 7
theories of origin, 7-9
Germinal epithelium, mitosis fre-
quency, 9
origin, 6
Gonadogenesis, 29-31
Gonads, embryogeny, 6
Grafts, gonad, 30, 31
Growth hormone, effects on ovogene-
sis, 24, 25
Heat treatment of eggs, 109
Hormones, ovarian, 2, 3
Hypertonicity, and activation, 109
Hypophysectomy, and ovogenesis,
22
Implantation, hormonal control of,
125 and ff.
time of, 124
Inner cell mass, 93
Lactation and delayed pregnancy in
rats and mice, 124 and ff.
Luteinizing hormone and ovogenesis,
26-28
Macacus, fertilizable life of ovum of,
88
159
160
SUBJECT INDEX
Oestrin, and sterilization, 120, 121
effect on cleavage and growth
stages, 117 and ff.
effect on egg cultures, 95, 122
effects on ovaries and ovogenesis,
25, 27
Oestrus cycle, 22
Oocytes, primate, 11
Ova, ovarian morphogenesis, 6 and ff.
ovarian, number at various ages,
23
primary, minimum size, 32
Ovariectomy, bilateral, effect on
ovaries, 15-20
effect on ovum growth, 116, 117
Ovaries, effects of x-ray on, 21
transplanted, 20, 21
Ovogenesis, effects of pituitary secre-
tions, 22 and ff.
Ovum, albumin covering in rabbit, 70
atresia and activation, 54, 55
binucleate, 57
condition at ovulation, 68
corona, 71
entry into uterus, 70 and ff.
fertilizable life, 82 and ff.
fragmentation, 52, 72 and ff.
human, recovery of, 63
maturation in rabbit, 46
methods of culture, 64-66
oestrin production, 44
polar body formation, 47-49
rate of passage in tubes, 74
recovery from tubes and uterus,
62-64
sizes in various classes of mam-
mals, 40
transplantation into oviduct, 66,
67, 94, 97
tubal, artificial activation of, 190
and ff.
tubal, cytology after activation,
107, 108
tubal, effect of age on behavior
in vitro, 106
Ovum — Continued
tubal, effect of retention in tubes,
111
unfertilized, behavior in vitro, 98
and ff.
unfertilized, tubal history of, 68
and ff.
vitellus at fertihzation, 80-82
Parthenogenesis, 1
and activation, 53 and ff.
ovarian, 57-59
Pituitary hormones, and delayed
pregnancy, 125-127
and maturation, 48-50, 56, 57
Pregnancy, and ovogenesis, 22
delayed, 124 and ff.
Pronucleus, 56, 110
Pseudomaturation, 33, 42, 43, 51
Pseudoparthenogenesis, 53
Rat, developmental stages, 11-14
Sperm, penetration into ovum, 82
swarm, 86
Suckling, and pseudopregnancy, 127
Superfetation, 50
Superovulation, 44
Tetrad formation in ovarian ova, 47
Tissue culture, 3, 64-66
Thyroidectomy and ovogenesis, 22
Thyroxin and maturation, 50, 51, 56,
57
Trephones, 28, 55
Trypsin, effect on ova, 79-81
Vesicle, germinal, 112 and ff.
X-rays, and pachytene stage of
meiosis, 39
effects on ovaries, 21
Zona pellucida, formation, 37
loss of, 1 16
